Abnormal meiosis in bisporic strains of white button mushroom Agaricus bisporus (Lange) imbach

A formerly developed method of microspreading of mushroom basidial nuclei was applied to study meiotic prophase I in bisporic white button mushroom (Agaricus bisporus) strains. Meiotic recombination and assemblage of axial structures (axial elements and synaptonemal complexes) of chromosomes in meiotic prophase I are interrelated. It is known that the frequency of meiotic recombination is reduced in the bisporic A. bisporus variety. We showed that formation of axial structures of meiotic chromosomes in bisporic strains of this mushroom was disrupted. The anomalous phenotypes in spread prophase nuclei are diverse. In leptotene and early zygotene, many nuclei contain abnormal, often short, and, as a rule, few chromosomal axial elements. The abnormalities in the formation of synaptonemal complexes at the zygotene-diplotene stage are of the same kind and even more pronounced. We discovered an important feature of meiosis in A. bisporus associated with fruit-body morphogenesis. Meiosis starting in basidia (meiocytes) of young closed fruit bodies is accompanied by disruption of chromatin condensation in prophase I and, probably, is arrested. After partial veil breakage, the course of meiosis normalizes. Preparations with clearly observable chromosomal axial structures can be obtained only at this stage of fruit-body development.     

Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis

Proper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei, but not that of a meiosis-specific Dmc1. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and/or multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphases I and II, suggesting the absence of DNA damage-induced cell cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase I.

     

A comparative study of male meiosis in Drosophila melanogaster and D. virilis

Male meiosis in D. melanogaster cytologically follows the usual pattern, whereas in D. melanogaster and in D. virilis oocytes the chromosomes clump into a karyosphere at early meiotic prophase and remain so up to metaphase I.Male meiosis in D. virilis spermatocytes has an intermediate character: a part of the chromatin clumps together in a karyosphere at early prophase, whereas the other part of the chromatin remains diffuse all through prophase. At the end of prophase, the diffuse chromatin becomes integrated into the karyosphere before metaphase I. During the meiotic divisions the chromosomes have the same clumped aspect as those in Drosophila oocytes and thus differ strikingly from the dividing chromosomes in D. melanogaster spermatocytes.In D. virilis spermatocytes the nucleolus exhibits changes during the meiotic prophase that may be related to synthetical activities. The DNA specific staining with the fluorochrome DAPI reveals the existence of extrachromosomal DNA in the later prophase. Other striking differences in meiotic events between the two Drosophila species concern the centrioles and spermiogenesis.     

The comparative effects of a mono- and a bi-functional alkylating agent on recombination inChlamydomonas reinhardi

Summary Synchronous populations of diploid cells ofChlamydomonas reinhardi were exposed to a bifunctional (Nitrogen Mustard) and a monofunctional (Diethyl Sulphate) alkylating agent at various stages of the pre-meiotic interphase and meiosis, and the effect on recombination in the arg 1/pab 2 region of linkage group I assessed. Both compounds affected recombination—the monofunctional one depressing it during two short periods, one just pre-meiosis when the main DNA duplication for meiosis is thought to occur, and the other in early prophase. The bifunctional compound on the other hand depressed recombination over an extended period. These effects have been related to the ability of the agents used to affect DNA synthesis and the different temporal patterns of response interpreted in terms of differential repair activities. The results provide additional evidence of the existence of two periods during which recombination can be affected.     

Abnormal meiosis in bisporic mutants of white button mushroom Agaricus bisporus (Lange) Imbach

A formerly developed method of obtaining spread preparations of mushroom basidial nuclei was applied to study of meiotic prophase I in bisporic white button mushroom (Agaricus bisporus) strains. Meiotic recombination and assemblage of axial structures (axial elements and synaptonemal complexes) of chromosomes in meiotic prophase I are interrelated. It is known that the frequency of meiotic recombination is reduced in the bisporic A. bisporus variety. We showed that formation of axial structures of meiotic chromosomes in bisporic strains of this mushroom was disrupted. The phenotypes of disruptions in spread prophase nuclei are diverse. In leptotene and early zygotene, many nuclei contain abnormal, often short, and, as a rule, few chromosomal axial elements. The abnormalities in the formation of synaptonemal complexes at the zygotene-diplotene stage are of the same kind and even more pronounced. We discovered an important feature of meiosis in A. bisporus associated with fruit-body morphogenesis. Meiosis starting in basidia (meiocytes) of young closed fruit bodies is accompanied by disruption of chromatin condensation in prophase I and, probably, is arrested. After indusium breakage, the course of meiosis normalizes. Preparations with clearly observable chromosomal axial structures can be obtained only at this stage of fruit-body development.     

Saccharomyces cerevisiae cells lacking the homologous pairing protein p175 SEP1 arrest at pachytene during meiotic prophase

Saccharomyces cerevisiae cells containing null mutations in the SEP1 gene, which encodes the homologous pairing and strand exchange protein p175 ^SEP1 enter pachytene with a delay. They arrest uniformly at this stage of meiotic prophase, probably revealing a checkpoint in the transition from pachytene to meiosis I. At the arrest point, the cells remain largely viable and are cytologically characterized by the duplicated but unseparated spindle pole bodies of equal size and by the persistence of the synaptonemal complex, a cytological marker for pachytene. In addition, fluorescence in situ hybridization revealed that in arrested mutant cells maximal chromatin condensation and normal homolog pairing is achieved, typical for pachytene in wild type. A hallmark of meiosis is the high level of homologous recombination, which was analyzed both genetically and physically. Formation and processing of the double-strand break intermediate in meiotic recombination is achieved prior to arrest. Physical intragenic (conversion) and intergenic (crossover) products are formed just prior to, or directly at, the arrest point. Structural deficits in synaptonemal complex morphology, failure to separate spindle pole bodies, and/or defects in prophase DNA metabolism might be responsible for triggering the observed arrest. The pachytene arrest in sep1 cells is likely to be regulatory, but is clearly different from the RAD9 checkpoint in meiotic prophase, which occurs prior to the pachytene stage.     

The<Emphasis Type="Italic">atspo11-1</Emphasis> mutation rescues <Emphasis Type="Italic">atxrcc3</Emphasis> meiotic chromosome fragmentation

Homologous recombination events occurring during meiotic prophase I ensure the proper segregation of homologous chromosomes at the first meiotic division. These events are initiated by programmed double-strand breaks produced by the Spo11 protein and repair of such breaks by homologous recombination requires a strand exchange activity provided by the Rad51 protein. We have recently reported that the absence of AtXrcc3, an ArabidopsisRad51 paralogue, leads to extensive chromosome fragmentation during meiosis, first visible in diplotene of meiotic prophase I. The present study clearly shows that this fragmentation results from un- or mis-repaired AtSpo11-1 induced double-strand breaks and is thus due to a specific defect in the meiotic recombination process.     

DNA polymerase mu interacts with a meiosis-specific RecA homolog Lim15 during meiosis in Coprinus cinereus

Meiosis is a fundamental process in eukaryotes. Homologous chromosomes are paired and recombined during meiotic prophase I, which results in variation among the gametes. However, the mechanism of recombination between the maternal and paternal chromosome is unknown. In this study, we report on the identification of interaction between Coprinus cinereus DNA polymerase mu (CcPol mu) and CcLim15/Dmc1, a meiosis-specific RecA-like protein, during meiosis. Interaction between these two proteins was confirmed using a GST-pull down assay. A two-hybrid assay revealed that the N-terminus of CcPol mu, which includes the BRCT domain, is responsible for binding the C-terminus of CcLim15. Furthermore, co-immunoprecipitation experiments indicate that these two proteins also interact in the crude extract of the meiotic cell. A significant proportion of CcPol mu and CcLim15 is shown to co-localize in nuclei from the leptotene/zygotene stage to the early pachytene stage during meiotic prophase I. Moreover, CcLim15 enhances polymerase activity of CcPol mu early in the reaction. These results suggest that CcPol mu might be recruited by CcLim15 and elongate the D-loop structure during homologous recombination in meiosis.     

Destruction of specific heterochromatic chromosomes during spermatogenesis in the Comstockiella chromosome system (Coccoidea: Homoptera)

In male coccids with the Comstockiella chromosome system, the set of chromosomes of paternal orgin becomes heterochromatic (H) during early cleavage. Just prior to prophase I of spermatogenesis, some of the H chromosomes are destroyed; the rest are eliminated following meiosis. In this report a Comstockiella sequence is described from Dactylopius opuntiae (2n=10) in which one chromosome pair is about three times longer than the others. During prophase I the number of small H chromosomes present varied from cyst to cyst, but the long H chromosome was present in every cyst. These observations provide the best evidence to date that in the Comstockiella system a particular chromosome may always escape destruction. An analysis of Kitchin's (1975) data about the frequency of prophase I cysts with 1–4 H chromosomes in three species of Parlatoria with 2n = 8 suggested that in these species chromosomes of similar size may have very different probabilities of being destroyed. Evidence that in other species with the Comstockiella system a particular H chromosome is always retained is reviewed, and the possibility that in Ancepaspis tridentata the variation in the length of the H chromosome retained is due to the partial destruction of the longest chromosome is discussed.     

Homolog pairing and sister chromatid cohesion in heterochromatin in <Emphasis Type="Italic">Drosophila</Emphasis> male meiosis I

Drosophila males undergo meiosis without recombination or chiasmata but homologous chromosomes pair and disjoin regularly. The X–Y pair utilizes a specific repeated sequence within the heterochromatic ribosomal DNA blocks as a pairing site. No pairing sites have yet been identified for the autosomes. To search for such sites, we utilized probes targeting specific heterochromatic regions to assay heterochromatin pairing sequences and behavior in meiosis by fluorescence in situ hybridization (FISH). We found that the small fourth chromosome pairs at heterochromatic region 61 and associates with the X chromosome throughout prophase I. Homolog pairing of the fourth chromosome is disrupted when the homolog conjunction complex is perturbed by mutations in SNM or MNM. On the other hand, six tested heterochromatic regions of the major autosomes proved to be largely unpaired after early prophase I, suggesting that stable homolog pairing sites do not exist in heterochromatin of the major autosomes. Furthermore, FISH analysis revealed two distinct patterns of sister chromatid cohesion in heterochromatin: regions with stable cohesion and regions lacking cohesion. This suggests that meiotic sister chromatid cohesion is incomplete within heterochromatin and may occur at specific preferential sites.     

Recombination correlates with synaptonemal complex length and chromatin loop size in bovids—insights into mammalian meiotic chromosomal organization

Homologous chromosomes exchange genetic information through recombination during meiosis, a process that increases genetic diversity, and is fundamental to sexual reproduction. In an attempt to shed light on the dynamics of mammalian recombination and its implications for genome organization, we have studied the recombination characteristics of 112 individuals belonging to 28 different species in the family Bovidae. In particular, we analyzed the distribution of RAD51 and MLH1 foci during the meiotic prophase I that serve, respectively, as proxies for double-strand breaks (DSBs) which form in early stages of meiosis and for crossovers. In addition, synaptonemal complex length and meiotic DNA loop size were estimated to explore how genome organization determines DSBs and crossover patterns. We show that although the number of meiotic DSBs per cell and recombination rates observed vary between individuals of the same species, these are correlated with diploid number as well as with synaptonemal complex and DNA loop sizes. Our results illustrate that genome packaging, DSB frequencies, and crossover rates tend to be correlated, while meiotic chromosomal axis length and DNA loop size are inversely correlated in mammals. Moreover, axis length, DSB frequency, and crossover frequencies all covary, suggesting that these correlations are established in the early stages of meiosis.     

The dsy10 Mutation of Arabidopsis results in desynapsis and a general breakdown in meiosis

The proper pairing and recombination of chromosomes during prophase is essential for the formation of gametes during meiosis. As part of studies to identify genes required for homologous chromosome pairing and recombination during meiosis in plants, we characterized a number of T-DNA-tagged, male-sterile mutants of Arabidopsis. Preliminary cytological studies on one line, 7219 which is male and female sterile, suggested that the mutation may disrupt meiosis and result in the formation of aberrant microsporocytes and microspores. In this report we present the results of a detailed analysis of meiosis in microsporocytes of sterile plants to elucidate the nature of the 7219 mutation. Analysis indicates that the mutation usually results in a desynaptic phenotype, with ten sister chromatids observed prior to metaphase I in most cells. Based on this, we named the mutation dsy10. The presence of several other meiotic defects suggests that dsy10 may not be a typical desynaptic mutant. Received: 15 December 2000 / Accepted: 19 April 2001     

Analysis of Polo-like kinase Cdc5 in the meiosis recombination checkpoint

In meiosis, accumulation of recombination intermediates or defects in chromosome synapsis trigger checkpoint-mediated arrest in prophase I. Such 'checkpoints' are important surveillance mechanisms that ensure temporal dependence of cell cycle events. The budding yeast Polo-like kinase, Cdc5, has been identified as a key regulator of the meiosis I chromosome segregation pattern. Here we have analysed the role of Cdc5 in the recombination checkpoint and observed that Polo-like kinase is not required for checkpoint activation in yeast meiosis. Surprisingly, depletion of CDC5 in the Drad17 checkpoint-defective background resulted in nuclear fragmentation to levels even higher than that observed inDdmc1 Drad17 cells that bypass the checkpoint arrest despite accumulating DNA double-strand breaks. The spindle morphology of Cdc5-depleted cells included short, thick metaphase I spindles in mononucleate cells and disassembled spindles in binucleate and tetranucleate cells, although this phenotype does not appear to be the cause of the nuclear fragmentation. An exaggeration of chromosome synapsis defects occurred in Cdc5-depleted Drad17 cells and may contribute to the nuclear fragmentation phenotype. The analysis also uncovered a role for Cdc5 in maintaining spindle integrity in Ddmc1 Drad17 cells. Further analysis confirmed that adaptation to DNA damage does occur in meiosis and that CDC5 is required for this process. The cdc5-ad mutation that renders cells unable to adapt to DNA damage in mitosis did not affect checkpoint adaptation in meiosis, indicating that the mechanisms of checkpoint adaptation in mitosis and meiosis are not fully conserved.     

The effects of inhibitors of RNA and protein synthesis on cytological development during meiosis

Summary An examination of the correlation between RNA and protein synthesis occurring during meiosis and cytological development was made in Trillium erectum microsporocytes. Various reagents known to act at various steps of protein biosynthesis were administered to cultured buds at different developmental stages with more or less effect depending on the stage rather than the reagent.Syntheses were found to be necessary for continued development of the microsporocytes during early prophase. Synthesis during meiotic prophase was also necessary for the maintainance of the condensed state of the late prophase chromosomes, the initial separation of the paired homologous chromosomes, and the orderly function of the spindle. Cytokinesis was readily disturbed at all treatment times. Pairing of homologous chromosomes was not affected and the prespecification of pairing is believed to occur at or near the time of DNA synthesis.The results indicate that the syntheses occurring during meiosis can be correlated with cytological developmental processes.Based on a thesis presented in partial fulfillment of the requirements for the Ph. D. degree at the University of Illinois, Department of Botany.     

ULTRASTRUCTURE OF MEIOSIS IN DASYA BAILLOUVIANA (RHODOPHYTA). I. PROPHASE I1

This first of two papers on ultrastructural observations of meiosis in the red alga Dasya baillouviana (Gmelin) Montague describes stages of prophase I of meiosis. Although the five stages of prophase were originally derived from light microscopic studies, the same stages were utilized for this study based on the developmental sequence of the synaptonemal complex, which has the same morphology and mode of development as those reported for other red algae. The cytoplasm in early prophase sporocytes was typically less electron dense than either vegetative cells or sporocytes in later stages of meiosis. The reduction in density suggests clearing of ribosomes and other cytoplasmic components prior to conversion from sporophyte to gametophyte control. Leptotene cells often had an amorphous, chromatin-free area, function unknown, which was not obviously associated with any specific nuclear region. Diplotene cells were characterized by nuclei containing prominent ring-shaped nucleoli composed of a dark staining ring of material surrounding an electron-translucent “vacuole.” Packets of electron-dense, fibrillar material were often noted in the cytoplasm of late prophase cells. These packets are thought to he “nuage,” a term applied to large cytoplasmic aggregations of RNA in germ cells of several other phyla. It is suggested that nuage may represent a new infusion of ribosomal and messenger RNA for post-meiotic development. The division pales are established by late prophase and a single polar ring is found within each large “exclusion zone” in close association with a pore-free area of nuclear envelope. Both annulate lamellae and small, numerous vesicles are located in the exclusion zones. The significance of the various aspects of prophase I is discussed with the overall observation that this phase of meiosis in red algae is very similar to the process in higher plant and animal cells.     

A cytological approach to studying meiotic recombination and chromosome dynamics in Arabidopsis thaliana male meiocytes in three dimensions

During meiotic prophase I chromosomes undergo dramatic conformational changes that accompany chromosome condensation, pairing and recombination between homologs. These changes include the anchoring of telomeres to the nuclear envelope and their clustering to form a bouquet. In plants, these events have been studied and illustrated in intact meiocytes of species with large genomes. Arabidopsis thaliana is an excellent genetic model in which major molecular pathways that control synapsis and recombination between homologs have been uncovered. Yet the study of chromosome dynamics is hampered by current cytological methods that disrupt the three‐dimensional (3D) architecture of the nucleus. Here we set up a protocol to preserve the 3D configuration of A. thaliana meiocytes. We showed that this technique is compatible with the use of a variety of antibodies that label structural and recombination proteins and were able to highlight the presence of clustered synapsis initiation centers at the nuclear periphery. By using fluorescence in situ hybridization we also studied the behavior of chromosomes during pre‐meiotic G₂ and prophase I, revealing the existence of a telomere bouquet during A. thaliana male meiosis. In addition we showed that the number of telomeres in a bouquet and its volume vary greatly, thus revealing the complexity of telomere behavior during meiotic prophase I. Finally, by using probes that label subtelomeric regions of individual chromosomes, we revealed differential localization behaviors of chromosome ends. Our protocol opens new areas of research for investigating chromosome dynamics in A. thaliana meiocytes.     

α-endosulfine (ENSA) regulates exit from prophase I arrest in mouse oocytes

Mammalian oocytes in ovarian follicles are arrested in meiosis at prophase I. This arrest is maintained until ovulation, upon which the oocyte exits from this arrest, progresses through meiosis I and to metaphase of meiosis II. The progression from prophase I to metaphase II, known as meiotic maturation, is mediated by signals that coordinate these transitions in the life of the oocyte. ENSA (α-endosulfine) and ARPP19 (cAMP-regulated phosphoprotein-19) have emerged as regulators of M-phase, with function in inhibition of protein phosphatase 2A (PP2A) activity. Inhibition of PP2A maintains the phosphorylated state of CDK1 substrates, thus allowing progression into and/or maintenance of an M-phase state. We show here ENSA in mouse oocytes plays a key role in the progression from prophase I arrest into M-phase of meiosis I. The majority of ENSA-deficient oocytes fail to exit from prophase I arrest. This function of ENSA in oocytes is dependent on PP2A, and specifically on the regulatory subunit PPP2R2D (also known as B55δ). Treatment of ENSA-deficient oocytes with Okadaic acid to inhibit PP2A rescues the defect in meiotic progression, with Okadaic acid-treated, ENSA-deficient oocytes being able to exit from prophase I arrest. Similarly, oocytes deficient in both ENSA and PPP2R2D are able to exit from prophase I arrest to an extent similar to wild-type oocytes. These data are evidence of a role for ENSA in regulating meiotic maturation in mammalian oocytes, and also have potential relevance to human oocyte biology, as mouse and human have genes encoding both Arpp19 and Ensa.     

BRCA2 deficiency in mice leads to meiotic impairment and infertility

The role of Brca2 in gametogenesis has been obscure because of embryonic lethality of the knockout mice. We generated Brca2-null mice carrying a human BAC with the BRCA2 gene. This construct rescues embryonic lethality and the mice develop normally. However, there is poor expression of the transgene in the gonads and the mice are infertile, allowing examination of the function of BRCA2 in gametogenesis. BRCA2-deficient spermatocytes fail to progress beyond the early prophase I stage of meiosis. Observations on localization of recombination-related and spermatogenic-related proteins suggest that the spermatocytes undergo early steps of recombination (DNA double strand break formation), but fail to complete recombination or initiate spermiogenic development. In contrast to the early meiotic prophase arrest of spermatocytes, some mutant oocytes can progress through meiotic prophase I, albeit with a high frequency of nuclear abnormalities, and can be fertilized and produce embryos. Nonetheless, there is marked depletion of germ cells in adult females. These studies provide evidence for key roles of the BRCA2 protein in mammalian gametogenesis and meiotic success.     

The Yeast Motor Protein, Kar3p, Is Essential for Meiosis I

The recognition and alignment of homologous chromosomes early in meiosis is essential for their subsequent segregation at anaphase I; however, the mechanism by which this occurs is unknown. We demonstrate here that, in the absence of the molecular motor, Kar3p, meiotic cells are blocked with prophase monopolar microtubule arrays and incomplete synaptonemal complex (SC) formation. kar3 mutants exhibit very low levels of heteroallelic recombination. kar3 mutants do produce double-strand breaks that act as initiation sites for meiotic recombination in yeast, but at levels severalfold reduced from wild-type. These data are consistent with a meiotic role for Kar3p in the events that culminate in synapsis of homologues.     

Extreme heterogeneity in the molecular events leading to the establishment of chiasmata during meiosis i in human oocytes

In humans, ~50% of conceptuses are chromosomally aneuploid as a consequence of errors in meiosis, and most of these aneuploid conceptuses result in spontaneous miscarriage. Of these aneuploidy events, 70% originate during maternal meiosis, with the majority proposed to arise as a direct result of defective crossing over during meiotic recombination in prophase I. By contrast, <1%-2% of mouse germ cells exhibit prophase I-related nondisjunction events. This disparity among mammalian species is surprising, given the conservation of genes and events that regulate meiotic progression. To understand the mechanisms that might be responsible for the high error rates seen in human females, we sought to further elucidate the regulation of meiotic prophase I at the molecular cytogenetic level. Given that these events occur during embryonic development in females, samples were obtained during a defined period of gestation (17-24 weeks). Here, we demonstrate that human oocytes enter meiotic prophase I and progress through early recombination events in a similar temporal framework to mice. However, at pachynema, when chromosomes are fully paired, we find significant heterogeneity in the localization of the MutL homologs, MLH1 and MLH3, among human oocyte populations. MLH1 and MLH3 have been shown to mark late-meiotic nodules that correlate well with--and are thought to give rise to--the sites of reciprocal recombination between homologous chromosomes, which suggests a possible 10-fold variation in the processing of nascent recombination events. If such variability persists through development and into adulthood, these data would suggest that as many as 30% of human oocytes are predisposed to aneuploidy as a result of prophase I defects in MutL homolog-related events.