End Of Pachytene Research Articles

Multiple reorganizations of the lateral elements of the synaptonemal complex facilitate homolog segregation in Bombyx mori oocytes.

Although Lepidopteran females build a synaptonemal complex (SC) in pachytene, homologs do not crossover, necessitating an alternative method of homolog conjunction. In Bombyx mori oocytes, the SC breaks down at the end of pachytene, and homolog associations are maintained by a large oocyte-specific structure, which we call the bivalent bridge (BB), connecting paired homologs. The BB is derived from at least some components of the SC lateral elements (LEs). It contains the HORMAD protein HOP1 and the LE protein SYCP2 and is formed by the fusion of the two LE derivatives. As diplotene progresses, the BB increases in width and acquires a layered structure with a thick band of HOP1 separating two layers of SYCP2. The HOP1 interacting protein, PCH2, joins the BB in mid-diplotene, and by late-diplotene, it lies in the middle of the HOP1 filament. This structure is maintained through metaphase I. SYCP2 and PCH2 are lost at anaphase I, and the BB no longer connects the separating homologs. However, a key component of the BB, HOP1, remains at the metaphase I plate. These changes in organization of the BB occur simultaneously with the movement of the kinetochore protein, DSN1, from within the BB at mid-diplotene to the edge of the homologs facing the poles by metaphase I. We view these data in context of models in which SC components and regulators can be repurposed to achieve different functions, a fascinating example of evolution achieving homolog conjunction in an alternative way with recycling of SC proteins.

Meiotic analyses show adaptations to maintenance of fertility in X1Y1X2Y2X3Y3X4Y4X5Y5 system of amazon frog Leptodactylus pentadactylus (Laurenti, 1768)

Heterozygous chromosomal rearrangements can result in failures during the meiotic cycle and the apoptosis of germline, making carrier individuals infertile. The Amazon frog Leptodactylus pentadactylus has a meiotic multivalent, composed of 12 sex chromosomes. The mechanisms by which this multi-chromosome system maintains fertility in males of this species remain undetermined. In this study we investigated the meiotic behavior of this multivalent to understand how synapse, recombination and epigenetic modifications contribute to maintaining fertility and chromosomal sexual determination in this species. Our sample had 2n = 22, with a ring formed by ten chromosomes in meiosis, indicating a new system of sex determination for this species (X1Y1X2Y2X3Y3X4Y4X5Y5). Synapsis occurs in the homologous terminal portion of the chromosomes, while part of the heterologous interstitial regions performed synaptic adjustment. The multivalent center remains asynaptic until the end of pachytene, with interlocks, gaps and rich-chromatin in histone H2A phosphorylation at serine 139 (γH2AX), suggesting transcriptional silence. In late pachytene, paired regions show repair of double strand-breaks (DSBs) with RAD51 homolog 1 (Rad51). These findings suggest that Rad51 persistence creates positive feedback at the pachytene checkpoint, allowing meiosis I to progress normally. Additionally, histone H3 trimethylation at lysine 27 in the pericentromeric heterochromatin of this anuran can suppress recombination in this region, preventing failed chromosomal segregation. Taken together, these results indicate that these meiotic adaptations are required for maintenance of fertility in L. pentadactylus.

Building bridges to move recombination complexes

A central feature of meiosis is pairing of homologous chromosomes, which occurs in two stages: coalignment of axes followed by installation of the synaptonemal complex (SC). Concomitantly, recombination complexes reposition from on-axis association to the SC central region. We show here that, in the fungus Sordaria macrospora, this critical transition is mediated by robust interaxis bridges that contain an axis component (Spo76/Pds5), DNA, plus colocalizing Mer3/Msh4 recombination proteins and the Zip2-Zip4 mediator complex. Mer3-Msh4-Zip2-Zip4 colocalizing foci are first released from their tight axis association, dependent on the SC transverse-filament protein Sme4/Zip1, before moving to bridges and thus to a between-axis position. Ensuing shortening of bridges and accompanying juxtaposition of axes to 100 nm enables installation of SC central elements at sites of between-axis Mer3-Msh4-Zip2-Zip4 complexes. We show also that the Zip2-Zip4 complex has an intrinsic affinity for chromosome axes at early leptotene, where it localizes independently of recombination, but is dependent on Mer3. Then, later, Zip2-Zip4 has an intrinsic affinity for the SC central element, where it ultimately localizes to sites of crossover complexes at the end of pachytene. These and other findings suggest that the fundamental role of Zip2-Zip4 is to mediate the recombination/structure interface at all post-double-strand break stages. We propose that Zip2-Zip4 directly mediates a molecular handoff of Mer3-Msh4 complexes, from association with axis components to association with SC central components, at the bridge stage, and then directly mediates central region installation during SC nucleation.

Cytogenetics of Aspidogaster limacoides (Trematoda, Aspidogastrea): karyotype, spermatocyte division, and genome size.

A detailed cytogenetic analysis of the aspidogastrean fluke Aspidogaster limacoides revealed a karyotype consisting of six medium-sized chromosome pairs. The first and the last pairs were two-armed while four remaining were one-armed; 2n = 12, n = 1 m + 1 m - sm + 4a. Fluorescence in situ hybridization with 18S ribosomal DNA (rDNA) probe detected a single cluster of ribosomal genes (NOR) located in pericentromeric regions of the long arms of the third chromosome pair in a site of secondary constriction apparent in meiotic prophase, especially in diplotene. The silver nitrate staining showed only a single active NOR site on one of homologous chromosomes in the majority of spermatogonia and spermatocyte divisions. A course of meiosis corresponded to standard schemes. The nucleolus was apparent in early meiotic spermatocytes and disintegrated by the end of pachytene. For the first time in Aspidogastrea, the genome size was determined. The flow cytometry showed 1.21 pg DNA per haploid nucleus in A. limacoides which is in accordance with relatively low genome sizes of other flukes and tapeworms (Neodermata). A comparison of cytogenetic data available to date in the fluke sister groups Aspidogastrea and Digenea suggests that the lower chromosome number of Aspidogastrea might represent an ancestral condition and their split might have been accompanied by an increase in chromosome number via either chromosome fissions or paleopolyploidy.

Rumors of Its Disassembly Have Been Greatly Exaggerated: The Secret Life of the Synaptonemal Complex at the Centromeres

Rumors of Its Disassembly Have Been Greatly Exaggerated: The Secret Life of the Synaptonemal Complex at the Centromeres

Error-prone ZW pairing and no evidence for meiotic sex chromosome inactivation in the chicken germ line.

In the male mouse the X and Y chromosomes pair and recombine within the small pseudoautosomal region. Genes located on the unsynapsed segments of the X and Y are transcriptionally silenced at pachytene by Meiotic Sex Chromosome Inactivation (MSCI). The degree to which MSCI is conserved in other vertebrates is currently unclear. In the female chicken the ZW bivalent is thought to undergo a transient phase of full synapsis at pachytene, starting from the homologous ends and spreading through the heterologous regions. It has been proposed that the repair of the ZW DNA double-strand breaks (DSBs) is postponed until diplotene and that the ZW bivalent is subject to MSCI, which is independent of its synaptic status. Here we present a distinct model of meiotic pairing and silencing of the ZW pair during chicken oogenesis. We show that, in most oocytes, DNA DSB foci on the ZW are resolved by the end of pachytene and that the ZW desynapses in broad synchrony with the autosomes. We unexpectedly find that ZW pairing is highly error prone, with many oocytes failing to engage in ZW synapsis and crossover formation. Oocytes with unsynapsed Z and W chromosomes nevertheless progress to the diplotene stage, suggesting that a checkpoint does not operate during pachytene in the chicken germ line. Using a combination of epigenetic profiling and RNA–FISH analysis, we find no evidence for MSCI, associated with neither the asynaptic ZW, as described in mammals, nor the synaptic ZW. The lack of conservation of MSCI in the chicken reopens the debate about the evolution of MSCI and its driving forces.

Synaptonemal Complex-Dependent Centromeric Clustering and the Initiation of Synapsis in Drosophila Oocytes

The pairing of homologous chromosomes and the intimate synapsis of the paired homologs by the synaptonemal complex (SC) are essential for subsequent meiotic processes including recombination and chromosome segregation. Here we show that the centromere clustering plays an important role in initiating homolog synapsis during meiosis in Drosophila females. Although centromeres are not clustered prior to the onset of meiosis, all four pairs of centromeres are actively clustered into one or two masses during early meiotic prophase. Within the 16-cell cyst, centromeric clustering appears to define the first step in the initiation of synapsis. Clustering is restricted to the nuclei that form the SC and is dependent on all known SC proteins. Surprisingly, both centromeric clusters and the SC components associated with them persist long after the disassembly of the euchromatic SC at the end of pachytene. The initiation of homologous recombination through the formation of programmed double-strand breaks (DSBs) is not required for either the formation or the maintenance of the centromeric clusters. Our data support a view in which the SC-mediated clustering at the centromeres is the initiating event for meiotic synapsis.

Nucleolar behavior during meiosis in four species of phyllostomid bats (Chiroptera, Mammalia)

We analyzed the behavior of the nucleolus, nucleolar structures and nucleolus organizer regions (NORs) during meiotic division in four species of phyllostomid bats that have different numbers and locations of NORs. Nucleoli began disassembly at leptotene, and the subcomponents released from the nucleolus were dispersed in the nucleoplasm, associated with perichromosomal regions, or they remained associated with NORs throughout division. In Phyllostomus discolor, a delay in nucleolus disassembly was observed; it disassembled by the end of pachytene. The RNA complexes identified by acridine orange staining were observed dispersed in the nucleoplasm and associated with perichromosomal regions. FISH with rDNA probe revealed the number of NORs of the species: one NOR in Carollia perspicillata, one pair in Platyrrhinus lineatus and P. discolor, and three pairs in Artibeus lituratus. During pachytene, there was a temporary dissociation of the homologous NORs, which returned to pairing at diplotene. The variation in the number (from one to three pairs) and location of NORs (in sex or autosomal chromosomes, at terminal or interstitial regions) did not seem to interfere with the nucleolar behavior of the different species because no variation in nucleolar behavior that could be correlated with the variation in the number and chromosomal location of NORs was detected.

A lampbrush phase in oocytes of Drosophila and its bearing upon mutagen sensitivity data

An autoradiographic study of RNA synthesis on S, oocyte chromosomes of D. melanogasler was performed together with a renewed light microscopic investigation of this stage. Corresponding oocyte stages inD. hydei and D.funebris were also studied. It is evident that the meiotic chromosomes of these species pass through a lampbrush phase at the end of pachytene. Transcriptions on both hetero- and euchromatin in the genome were confirmed during the lampbrush phase in D. melanogasler. The lampbrush phase seems to parallel a decreased mutation sensitivity known to occur during this stage of oocyte development. Oocytes of the more mutation sensitive D. melanogasler mutant c(3)G also pass through a lampbrush phase.

The mechanism of secondary nondisjunction in Drosophila melanogaster females.

Bridges (1916) observed that X chromosome nondisjunction was much more frequent in XXY females than it was in genetically normal XX females. In addition, virtually all cases of X nondisjunction in XXY females were due to XX <--> Y segregational events in oocytes in which the two X chromosomes had failed to undergo crossing over. He referred to these XX <--> Y segregation events as "secondary nondisjunction." Cooper (1948) proposed that secondary nondisjunction results from the formation of an X-Y-X trivalent, such that the Y chromosome directs the segregation of two achiasmate X chromosomes to opposite poles on the first meiotic spindle. Using in situ hybridization to X and YL chromosomal satellite sequences, we demonstrate that XX <--> Y segregations are indeed presaged by physical associations of the X and Y chromosomal heterochromatin. The physical colocalization of the three sex chromosomes is observed in virtually all oocytes in early prophase and maintained at high frequency until midprophase in all genotypes examined. Although these XXY associations are usually dissolved by late prophase in oocytes that undergo X chromosomal crossing over, they are maintained throughout prophase in oocytes with nonexchange X chromosomes. The persistence of such XXY associations in the absence of exchange presumably facilitates the segregation of the two X chromosomes and the Y chromosome to opposite poles on the developing meiotic spindle. Moreover, the observation that XXY pairings are dissolved at the end of pachytene in oocytes that do undergo X chromosomal crossing over demonstrates that exchanges can alter heterochromatic (and thus presumably centromeric) associations during meiotic prophase.

Ovogenèse et transmission des bactéries symbiotiques chez le charançon Sitophilus oryzae L. (Coleoptera: Curculionoidea)

Summary Oogenesis and transmission of symbiotic bacteria in the weevil Sitophilus oryzae L. (Coleoptera: Dryophthoridae). Among Coleoptera, and particularly in numerous Dryophthoridae, the insects share intracellular symbiosis with hereditary Gram-negative bacteria (or endocytobiotes). Sitophilus oryzae Primary Endosymbiote (SOPE) belongs to the γ3-proteobacteria group. It is a pleomorphic rod shaped (1 to 30 μm ), non-sporulating and non-ciliated bacterium. In the bacteriocyte the bacteria are not included in vacuoles but lie freely in the cytoplasm. SOPE is closely related to Escherichia coli, with 95% homology on the 16S rDNA gene, and also the primary endosymbiotes of Sitophilus zeamais (97,8% identity). In addition to SOPE, several wild strains of S. oryzae harbor a second symbiotic bacterium, that belongs to the B-group of Wolbachia (an α-proteobacterium). They are not considered in this work since studied strains were deprived of Wolbachia. Previous studies have shown that SOPE is transmitted to th...

Temporal progression of recombination in human males

To date, immunocytology has been used in humans to detect a limited number of meiotic proteins: components of the synaptonemal complex (SCP1 and SCP3) and some proteins known to participate in recombination events, such as MLH1 or RAD51. However, the colocalization or coexistence of proteins known to participate during the different stages of human meiosis remains largely unstudied, and these studies could provide important clues about the mechanics of recombination. This work reports the relative timing and localization of five different meiotic proteins that have previously been implicated in human homologous recombination [RAD51, replication protein A (RPA), MSH4, MLH1 and MLH3]. MSH4 foci appear concurrently with synapsis initiation at zygotene, shortly after the first RAD51 foci are detected. The presence of RPA in MSH4 foci was noted, suggesting that these two proteins may act co-operatively. Both RPA and MSH4 foci reach maximal numbers at the end of zygotene, when synapsis is concluding. From this point, RPA foci all but disappear by the end of pachytene, whereas MSH4 foci decline to a stable number at mid-pachytene, where they localize with MLH1/MLH3 recombination sites. We discuss a possible role for MSH4 in synapsis initiation and/or maintenance.

The Synaptonemal Complex Complement of the Wax Moth, Galleria Mellonella

Synaptonemal complexes (SCs) of pachytene bivalents from oocytes and spermatocytes of the wax moth, Galleria mellonella, were studied with a microspreading technique. In both sexes 30 bivalents were found. Male SCs in contrast to female SCs contained recombination nodules. No specific bivalent could be recognized in male complements. In female complements, the WZ bivalent was recognized among the three longest bivalents by its delayed pairing and/or unequal lengths of the lateral elements. Pairing of WZ bivalents is complete and lateral elements are nearly equalized in a few cells, presumably at the end of pachytene.

Synaptonemal Complex Formation in Spermatocytes of the Autotriploid Rainbow Trout, Oncorhynchus Mykiss (Pisces, Salmonidae)

Meiotic cells of triploid male rainbow trouts were analyzed by a surface-spreading SC technique in order to show the process of chromosome synapsis. At zygotene the formation of SCs involved, almost exclusively, two sets of lateral elements (LEs), and the remaining set of LEs presented different synaptic configurations involving one, two, three, or four LEs. The absence of SCs involving more than two Les from mid- to late pachytene indicates that the multivalents produced by synapsis involving more than two LEs, are eliminated before the end of pachytene; thus, the mechanism of chromosome pairing in triploid male rainbow trouts produces, almost exclusively, bivalents with a probable extensive nonhomologous synapsis involving the extra set of chromosomes.

A deficiency screen of the major autosomes identifies a gene (matrimony) that is haplo-insufficient for achiasmate segregation in Drosophila oocytes.

In Drosophila oocytes, euchromatic homolog-homolog associations are released at the end of pachytene, while heterochromatic pairings persist until metaphase I. A screen of 123 autosomal deficiencies for dominant effects on achiasmate chromosome segregation has identified a single gene that is haplo-insufficient for homologous achiasmate segregation and whose product may be required for the maintenance of such heterochromatic pairings. Of the deficiencies tested, only one exhibited a strong dominant effect on achiasmate segregation, inducing both X and fourth chromosome nondisjunction in FM7/X females. Five overlapping deficiencies showed a similar dominant effect on achiasmate chromosome disjunction and mapped the haplo-insufficient meiotic gene to a small interval within 66C7-12. A P-element insertion mutation in this interval exhibits a similar dominant effect on achiasmate segregation, inducing both high levels of X and fourth chromosome nondisjunction in FM7/X females and high levels of fourth chromosome nondisjunction in X/X females. The insertion site for this P element lies immediately upstream of CG18543, and germline expression of a UAS-CG18543 cDNA construct driven by nanos-GAL4 fully rescues the dominant meiotic defect. We conclude that CG18543 is the haplo-insufficient gene and have renamed this gene matrimony (mtrm). Cytological studies of prometaphase and metaphase I in mtrm hemizygotes demonstrate that achiasmate chromosomes are not properly positioned with respect to their homolog on the meiotic spindle. One possible, albeit speculative, interpretation of these data is that the presence of only a single copy of mtrm disrupts the function of whatever "glue" holds heterochromatically paired homologs together from the end of pachytene until metaphase I.

Ultrastructure, meiotic behavior, and evolution of sex chromosomes of the genus Ellobius

The whole-mount SC preparations from males of three species of the genus Ellobius (Ellobius fuscocapillus, Ellobius lutescens), and Ellobius tancrei1 were studied by electron microscopy. In the males of Ellobius fuscocapillus, behavioral peculiarities of the sex bivalent (viz. the normal male heterozygosity) are characterized by early complete desynapsis of sex chromosomes (X, Y), occurring at late pachytene-early diplotene. The karyotype of species Ellobius lutescens is unique for mammals. In both sexes it is characterized by an odd number of chromosomes (2n=17). At prophase I the unpaired chromosome 9 is not involved in synapsis with other chromosomes and forms a sex body at the end of pachytene.

The cellular program for the formation and dissolution of the synaptonemal complex in Coprinus.

Inhibition of protein synthesis by cycloheximide on processes in meiosis was used to probe the cellular program for the formation and dissolution of the synaptonemal complex (SC) in the synchronous meiotic system of Coprinus cinereus. The pathway for the synthesis and assembly of the synaptonemal complex is proposed to be as follows: (1) synthesis and assembly of lateral components on the chromosomes; (2) synthesis and assembly of the central components in the nucleolus; (3) the lateral components of the homologous chromosomes are brought together to pair when the homologous pairing occurs at zygotene; (4) the transport of the central components from the nucleolus to join the paired lateral components and thus complete the synaptonemal complex. Continued protein synthesis is required for all steps. Step (1) is nearly complete 2 h after the onset of karyogamy, because continued assembly is possible in the presence of cycloheximide. The transition point for step (2) is 4 h after the onset of karyogamy, as inhibition at this point results in accumulation of central components in the nucleolar dense body. The paired lateral components of step (3) are deprived of the central component. The transition point for step (4) is 5 h after the onset of karyogamy, for inhibition at this point no longer prevents transport. Two steps are proposed for dissociation and dissolution of the SC at the end of pachytene. Protein synthesis is required for the dissolution of SC. Inhibition at this point causes accumulation of polycomplexes. Mutations in various organisms from the literature relating to the SC support the validity of the proposed pathway.

Human meiosis V. Substages of pachytene in human spermatogenesis

The pachytene stage of human spermatogenesis has been investigated by serial sectioning of spermatocytes and three dimensional reconstruction of nuclei from electron micrographs. The analysis of 73 completely reconstructed normal nuclei from late zygotene to early diplotene revealed a continuous sequence of changes in the fine structure and morphology. The developmental sequence permits the recognition of 7 substages on the basis of morphological changes of autosomal chromatin, centromeric, chromatin, the XY bivalent, the secondary constriction on bivalents 1,9 and 16, the nucleolus and the centrioles. Of particular interest is the observation that the disappearance of the synaptonemal complex segment between the X and Y chromosomes at mid pachytene is only temporary, a distinct complex segment being present in nearly all analysed XY bivalents by the end of pachytene. The number of recombination nodules and recombination bars in nuclei of the different substages is recorded. The total number of nodules decreases from 75 at the beginning of pachytene to 1 or none at the end of this state. The bars appear at early pachytene, reach a number of 35 at mid pachytene and disappear following the nodules towards the end of pachytene. The morphology of pachytene nuclei at various stages of degeneration as well as the structural characteristics of two tetraploid spermatocytes are described.

Le caryotype et la spermatog�n�se de Mus setulosus (bandes Q, C, G et coloration argentique)

The karyotype and the spermatogenesis of Mus setulosus (G, Q, C bands and Ag-staining). — The G, Q and C banding patterns of Mus setulosus are described. In the male five Ag-NORs were detectable, located at the distal extremities of the chromosomes. The meiotic divisions of Mus setulosus ♂ were studied by silver-staining and C-banding. The X and Y chromosomes remain close to each other during leptotene, zygotene and pachytene; they do not establish and end-to-end or side-by-side association. During diplotene, diakinesis and metaphase I, they most often are separated. At pachytene the bivalents 5 and 7 lie near each other and form a ‘heterochromatic vesicle’. The r-RNA synthesis declines at the end of pachytene and stops during diplotene, diakinesis, metaphase I and II. During these four stages the centromeres are Ag-positive. A comparative analysis of G-bands revealed a great number of analogies between Mus musculus and M. setulosus, which confirms the hypothesis that M. setulosus originated from a Mus of the musculus group. Nevertheless the existence of non-homologous pairs and the differences in the distribution and the size of heterochromatic blocks show that the derivation of the karyotype of setulosus from that of musculus imply several complex rearrangements, the nature of which could not be determined despite the refinement of the actual techniques.

Chromosome pairing, recombination nodules and chiasma formation in diploid Bombyx males

Serial sectioning and three dimensional reconstruction of 82 nuclei divided into 15 well characterized substages covering the period from early zygotene to metaphase I in diploid Bombyx spermatocytes have permitted the following observations and conclusions: 1) At late zygotene, a mean of four chromosome and bivalent interlockings was found per nucleus. 2) During the transition from zygotene to pachytene, all interlockings resolve by breakage and reunion of chromosomes or bivalents. 3) Recombination nodules first appear at early zygotene, reach a maximum of 91 per nucleus by late zygotene, decrease to 55 per nucleus at mid pachytene and increase again to 70–75 towards the end of pachytene. 4) At late zýgotene and pachytene, nodules are essentially distributed at random among bivalents as well as along bivalents. 5) Small, but important deviations from randomness were, however, revealed: Three percent of the bivalents were without nodules at late zygotene, 8% at early pachytene but at mid and late pachytene, only 2% were devoid of nodules. 6) From mid pachytene, an increasing fraction of the recombination nodules becomes larger and more irregular in shape. These are termed chromatin nodules and at the pachytene-diplotene transition, nearly all nodules are of this type. 7) During early diplotene, the bulk of the chromatin decondenses leaving only about 60 major condensed domains. These domains originate from chromatin nodules and are frequently associated with a piece of synaptonemal complex which in its central region contains a dense core resembling a recombination nodule. 8) At late diplotene, the condensed domains of the bivalents consist of two dense chromatin segments, one on each of the homologues, and bridged by a complex circular structure giving the regions a characteristic tripartite appearance. 9) At mid diakinesis, the circular structures are located in chromatin bridges — chiasmata —between the homologous chromosomes. 10) The number of chiasmata per nucleus is constant from mid diplotene to mid diakinesis and is distributed among the bivalents similar to the distribution of recombination nodules at mid and late pachytene. 11) The circular chiasma components are eliminated from late diakinesis until metaphase I. 12) Finally, each metaphase I bivalent contains four distinct localized centromeres, two by two facing opposite spindle poles.