A pathway for synapsis initiation during zygotene in Drosophila oocytes

Formation of the synaptonemal complex (SC), or synapsis, between homologs in meiosis is essential for crossing over and chromosome segregation [1-4]. How SC assembly initiates is poorly understood but may have a critical role in ensuring synapsis between homologs and regulating double-strand break (DSB) and crossover formation. We investigated the genetic requirements for synapsis in Drosophila and found that there are three temporally and genetically distinct stages of synapsis initiation. In "early zygotene" oocytes, synapsis is only observed at the centromeres. We also found that nonhomologous centromeres are clustered during this process. In "mid-zygotene" oocytes, SC initiates at several euchromatic sites. The centromeric and first euchromatic SC initiation sites depend on the cohesion protein ORD. In "late zygotene" oocytes, SC initiates at many more sites that depend on the Kleisin-like protein C(2)M. Surprisingly, late zygotene synapsis initiation events are independent of the earlier mid-zygotene events, whereas both mid and late synapsis initiation events depend on the cohesin subunits SMC1 and SMC3. We propose that the enrichment of cohesion proteins at specific sites promotes homolog interactions and the initiation of euchromatic SC assembly independent of DSBs. Furthermore, the early euchromatic SC initiation events at mid-zygotene may be required for DSBs to be repaired as crossovers.     

The initiation of homologous chromosome synapsis in mouse fetal oocytes is not directly driven by centromere and telomere clustering in the bouquet

We investigated the behaviour of centromeres and distal telomeres during the initial phases of female meiosis in mice. In particular, we wished to determine whether clustering of centromeres and telomeres (bouquet formation) played the same crucial role in homologous chromosome pairing in female meiosis as it does in the male. We found that synapsis (intimate homologous chromosome pairing) is most frequently initiated in the interstitial regions of homologous chromosomes, apparently ahead of the distal regions. The proximal ends of the chromosomes appear to be disfavoured for synaptic initiation. Moreover, initiation of synapsis occurred in oocytes that showed little or no evidence of bouquet formation. A bouquet was present in a substantial proportion of cells at mid to late zygotene, and was still present in some pachytene oocytes. This pattern of bouquet formation and pairing initiation is in stark contrast to that previously described in the male mouse. We propose that although dynamic movements of centromeres and telomeres to form clusters may facilitate alignment of homologues or homologous chromosome segments during zygotene, in the female mouse positional control of synaptic initiation is dependent on some other mechanism.     

Synaptonemal polycomplexes in Drosophila melanogaster

Synaptonemal polycomplexes are described for the first time in Drosophila melanogaster females. They were found in apparently degenerating 16-cell clusters in germarial region two of females heterozygous for the c(3)G ¹⁷ mutation. The remaining 16-cell clusters in the germaria were normal in appearance. In a single case a polycomplex was found in the cytoplasm, presumably due to the breakdown of the nuclear envelope. In all other cases, the polycomplexes were closely associated with the nucleolus. The role of the nucleolus in the formation of polycomplexes as well as the possibility that these polycomplexes are normal features of the meiotic prophase in Drosophila are discussed.     

Sex-specific telomere redistribution and synapsis initiation in cattle oogenesis

The process of homolog pairing is well characterised in meiosis of male mammals, but much less information is available from female meiosis. We have therefore studied telomere dynamics by FISH and synapsis formation by immunostaining of synaptonemal complex proteins (SCP3, SCP1) on ovarian sections from 15 bovine fetuses, which covered the entire female prophase I. Telomeres displayed a dispersed intranuclear distribution in oogonia and relocated to the nuclear periphery during the preleptotene stage. Tight telomere clustering (bouquet formation) coincided with synapsis initiation at the leptotene/zygotene transition. Clustering of telomeres persisted during zygotene and even into the pachytene stage in a subset of nuclei, while it was absent in diplotene/dictyotene stage nuclei. Thus, the bouquet stage in the bovine female lasts significantly longer than in the male. Further, we observed that synapsis in the female initiated both terminally and interstitially in earliest zygotene stage oocytes, which contrasts with the predominantly terminal synapsis initiation in early zygotene spermatocytes of the bovine male. Altogether, our data disclose a sex-specific difference in telomere dynamics and synapsis initiation patterns in male and female bovine germ cells that may be related to the sex-specific differences in recombination rates observed in this and other mammalian species.     

Gene conversion, recombination nodules, and the initiation of meiotic synapsis

The nature of the relationship between the two types of meiotic recombination outcomes, exchange (crossing-over) and simple gene conversion, has been debated for years. I here propose that these two types of events are not necessarily causally related and hypothesize that the primary role of events detected as simple gene conversion is in the recognition of homology during synapsis.     

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.     

Tetrameric structure of centromeric nucleosomes in interphase Drosophila cells

Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3). However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical “beads-on-a-string” appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity.     

Imposition of crossover interference through the nonrandom distribution of synapsis initiation complexes

Meiotic crossovers (COs) are nonrandomly distributed along chromosomes such that two COs seldom occur close together, a phenomenon known as CO interference. We have used genetic and cytological methods to investigate interference mechanisms in budding yeast. Assembly of the synaptonemal complex (SC) initiates at a few sites along each chromosome, triggered by a complex of proteins (including Zip2 and Zip3) called the synapsis initiation complex (SIC). We found that SICs, like COs, display interference, supporting the hypothesis that COs occur at synapsis initiation sites. Unexpectedly, we found that SICs show interference in mutants in which CO interference is abolished; one explanation is that these same mutations eliminate the subset of COs that normally occur at SICs. Since SICs are assembled in advance of SC and they are properly positioned even in the absence of SC formation, these data clearly demonstrate an aspect of interference that is independent of synapsis.     

Somatic synapsis of eu- and heterochromatic regions of Drosophila melanogaster chromosomes

Quantitative cytogenetical analysis has been used to study the synapsis of D. melanogaster neuroblast mitotic chromosomes from normal females, flies with heterozygous deletions, duplications or inversions in the heterochromatic regions of chromosome 2 and in triploid females. In all these genotypes chromocentric fusion of heterochromatic regions of heterologous chromosomes is observed. Eu- and heterochromatic regions of homologous chromosomes are intimately paired at the same time during the cell cycle. The structural rearrangements lead to reduced frequencies of chromocentric association as well as of homologous synapsis compared with the frequencies in the wild-type. The results obtained are discussed with respect to the general problem of the homologous interaction of chromosomes and the significance of heterochromatin for these processes.     

The pattern of polytene chromosome synapsis in Drosophila species and interspecific hybrids

The pairing of polytene chromosomes was investigated in Drosophila melanogaster, Drosophila simulans and their hybrids as well as in species of the D. virilis group and in F₁ hybrids between the species of this group. The study of frequency and extent of asynapsis revealed non-random distribution along chromosome arms both in interspecific hybrids and pure Drosophila species. It is suggested that definite chromosome regions exhibiting high pairing frequency serve as initiation sites of synapsis in salivary gland chromosomes.     

Unified mode of centromeric protection by shugoshin in mammalian oocytes and somatic cells

Reductional chromosome segregation in germ cells, where sister chromatids are pulled to the same pole, accompanies the protection of cohesin at centromeres from separase cleavage. Here, we show that mammalian shugoshin Sgo2 is expressed in germ cells and is solely responsible for the centromeric localization of PP2A and the protection of cohesin Rec8 in oocytes, proving conservation of the mechanism from yeast to mammals. However, this role of Sgo2 contrasts with its mitotic role in protecting centromeric cohesin only from prophase dissociation, but never from anaphase cleavage. We demonstrate that, in somatic cells, shugoshin colocalizes with cohesin in prophase or prometaphase, but their localizations become separate when centromeres are pulled oppositely at metaphase. Remarkably, if tension is artificially removed from the centromeres at the metaphase-anaphase transition, cohesin at the centromeres can be protected from separase cleavage even in somatic cells, as in germ cells. These results argue for a unified view of centromeric protection by shugoshin in mitosis and meiosis.     

Assembly of Drosophila centromeric chromatin proteins during mitosis

Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. We analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and CAL1, which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation.     

Assembly of Drosophila centromeric nucleosomes requires CID dimerization

Centromeres are essential chromosomal regions required for kinetochore assembly and chromosome segregation. The composition and organization of centromeric nucleosomes containing the essential histone H3 variant CENP-A (CID in Drosophila) is a fundamental, unresolved issue. Using immunoprecipitation of CID mononucleosomes and cysteine crosslinking, we demonstrate that centromeric nucleosomes contain CID dimers in vivo. Furthermore, CID dimerization and centromeric targeting require a residue implicated in formation of the four-helix bundle, which mediates intranucleosomal H3 dimerization and nucleosome integrity. Taken together, our findings suggest that CID nucleosomes are octameric in vivo and that CID dimerization is essential for correct centromere assembly.     

Prdm9 deficiency of rat oocytes causes synapsis among non-homologous chromosomes and aneuploidy

Mammalian Genome - Aneuploidy (abnormal chromosome number) accompanies reduced ovarian function in humans and mice, but the reasons behind this concomitance remain underexplored. Some variants in...     

Cap-independent translation initiation in Xenopus oocytes.

Eukaryotic cellular mRNAs contain a cap at their 5'-ends, but some viral and cellular mRNAs bypass the cap-dependent mechanism of translation initiation in favor of internal entry of ribosomes at specific RNA sequences. Cap-dependent initiation requires intact initiation factor eIF4G (formerly eIF-4gamma, eIF-4Fgamma or p220), whereas internal initiation can proceed with eIF4G cleaved by picornaviral 2A or L proteases. Injection of recombinant coxsackievirus B4 protease 2A into Xenopus oocytes led to complete cleavage of endogenous eIF4G, but protein synthesis decreased by only 35%. Co-injection of edeine reduced synthesis by >90%, indicating that eIF4G-independent synthesis involved ongoing initiation. The spectrum of endogenous proteins synthesized was very similar in the presence or absence of intact eIF4G. Translation of exogenous rabbit globin mRNA, by contrast, was drastically inhibited by eIF4G cleavage. The N-terminal cleavage product of eIF4G (cpN), which binds eIF4E, was completely degraded within 6-12 h, while the C-terminal cleavage product (cpC), which binds to eIF3 and eIF4A, was more stable over the same period. Thus, translation initiation of most endogenous mRNAs inXenopusoocytes requires no eIF4G, or perhaps only cpC, suggesting a cap-independent mechanism.     

Pericentromeric chromatin reorganisation follows the initiation of recombination and coincides with early events of synapsis in cereals

The reciprocal exchange of genetic information between homologous chromosomes during meiotic recombination is essential to secure balanced chromosome segregation and to promote genetic diversity. The chromosomal position and frequency of reciprocal genetic exchange shapes the efficiency of breeding programmes and influences crop improvement under a changing climate. In large genome cereals, such as wheat and barley, crossovers are consistently restricted to subtelomeric chromosomal regions, thus preventing favourable allele combinations being formed within a considerable proportion of the genome, including interstitial and pericentromeric chromatin. Understanding the key elements driving crossover designation is therefore essential to broaden the regions available for crossovers. Here, we followed early meiotic chromatin dynamism in cereals through the visualisation of a homologous barley chromosome arm pair stably transferred into the wheat genetic background. By capturing the dynamics of a single chromosome arm at the same time as detecting the undergoing events of meiotic recombination and synapsis, we showed that subtelomeric chromatin of homologues synchronously transitions to an open chromatin structure during recombination initiation. By contrast, pericentromeric and interstitial regions preserved their closed chromatin organisation and become unpackaged only later, concomitant with initiation of recombinatorial repair and the initial assembly of the synaptonemal complex. Our results raise the possibility that the closed pericentromeric chromatin structure in cereals may influence the fate decision during recombination initiation, as well as the spatial development of synapsis, and may also explain the suppression of crossover events in the proximity of the centromeres.     

RNA sorting in Drosophila oocytes and embryos.

Many RNAs involved in determination of the oocyte, specification of embryonic axes, and establishment of germ cells in Drosophila are localized asymmetrically within the developing egg or syncytial embryo. Here I review the current state of knowledge about the cis-acting sequences involved in RNA targeting, RNA binding proteins; gene activities implicated in localizing specific RNAs, and the role of the tubulin and actin cytoskeletons in RNA sorting within the oocyte. Targeted RNAs are often under complex translational control, and the translational control of two RNAs that localize to the posterior of the oocyte, oskar and nanos, is also discussed. Prospects for filling gaps in our knowledge about the mechanisms of localizing RNAs and the importance of RNA sorting in regulating gene expression are also explored.     

Synaptonemal complex analysis of spermatocytes of Talpa occidentalis (Insectivora, Mammalia): autosomal synapsis and substaging of zygonema and pachynema

Spermatocytes from the mole, Talpa occidentalis, a species that includes both XX males and intersexes, were surface-spread and silver-stained to substage meiotic prophase from early zygonema through pachynema. In zygonema, only the Z2 and Z3 substages were found. This stage differed in comparison with such species as the Chinese hamster, laboratory mouse, and deer mouse, which belong to orders other than Insectivora. Pachynema, in which five substages were established (P1-P5), seems to be a more homogeneous stage, and remarkable differences with respect to the above-mentioned species were not found. Synaptic adjustment was demonstrated in X-Y pairing. Nonhomologous pairing was evident at the Y-centromeric region and considered likely in the proximal arm of this chromosome. In addition to sequencing the events taking place during zygonema and pachynema in males from a wild population in which some members show sex reversal, our finding represents the first attempt to substage zygonema and pachynema in an Insectivore species, thus contributing to current knowledge of the nature and degree of variability in the mammalian synaptic process.