Cohesin component dynamics during meiotic prophase I in mammalian oocytes

Cohesins are chromosomal proteins that form complexes involved in the maintenance of sister chromatid cohesion during division of somatic and germ cells. Three meiosis-specific cohesin subunits have been reported in mammals, REC8, STAG3 and SMC1 beta; their expression in mouse spermatocytes has also been described. Here we studied the localization of different meiotic and mitotic cohesin components during prophase I in human and murine female germ cells. In normal and atretic human fetal oocytes, from leptotene to diplotene stages, REC8 and STAG3 colocalize in fibers. In murine oocytes, SMC1beta, SMC3 and STAG3 are localized along fibers that correspond first to the chromosome axis and then to the synaptonemal complex in pachytene. Mitotic cohesin subunit RAD21 is also found in fibers that decorate the SC during prophase I in mouse oocytes, suggesting a role for this cohesin in mammalian sister chromatid cohesion in female meiosis. We observed that, unlike human oocytes, murine synaptonemal complex protein SYCP3 localizes to nucleoli throughout prophase I stages, and centromeres cluster in discrete locations from leptotene to dictyate. At difference from meiosis in male mice, the cohesin axis is progressively lost during the first week after birth in females with a parallel destruction of the axial elements at dictyate arrest, demonstrating sexual dimorphism in sister chromatid cohesion in meiosis.     

Lampbrush chromosomes enable study of cohesin dynamics

The lampbrush chromosomes present in the nuclei of amphibian oocytes offer unique biological approaches for study of the mechanisms that regulate chromatin structure with high spatial resolution. We discuss fundamental aspects of the remarkable organization and plasticity exhibited by lampbrush chromosomes. We then utilize lampbrush chromosomes to characterize the chromosomal distribution and dynamics of cohesin, the four-protein complex (RAD21/MCD1/SCC1, SMC1, SMC3, SCC3/SA2) responsible for sister chromatid cohesion. We find that endogenous SMC3 and newly expressed hRAD21 co-localize on chromosomal axes, sites where sister chromatids are tightly paired. We present evidence suggesting that hRAD21 recruitment to lampbrush chromosomes is modulated by chromosomal SMC1 and SMC3. Notably, using a technique for de novo chromosome assembly, we demonstrate that both SMC3 and hRAD21 are recruited to single, unreplicated lampbrush chromatids. Finally, we used our novel method of analyzing the oocyte nucleus under oil combined with fluorescence recovery after photobleaching, to provide direct evidence that cohesin is highly dynamic at discrete, condensed chromosomal regions. Collectively, these data demonstrate that lampbrush chromosomes provide a unique and powerful tool for combining biochemical and cytological analyses for dissection of complex chromosomal processes.     

Cohesin proteins load sequentially during prophase I in tomato primary microsporocytes

Proteins of the cohesin complex are essential for sister chromatid cohesion and proper chromosome segregation during both mitosis and meiosis. Cohesin proteins are also components of axial elements/lateral elements (AE/LEs) of synaptonemal complexes (SCs) during meiosis, and cohesins are thought to play an important role in meiotic chromosome morphogenesis and recombination. Here, we have examined the cytological behavior of four cohesin proteins (SMC1, SMC3, SCC3, and REC8/SYN1) during early prophase I in tomato microsporocytes using immunolabeling. All four cohesins are discontinuously distributed along the length of AE/LEs from leptotene through early diplotene. Based on current models for the cohesin complex, the four cohesin proteins should be present at the same time and place in equivalent amounts. However, we observed that cohesins often do not colocalize at the same AE/LE positions, and cohesins differ in when they load onto and dissociate from AE/LEs of early prophase I chromosomes. Cohesin labeling of LEs from pachytene nuclei is similar through euchromatin, pericentric heterochromatin, and kinetochores but is distinctly reduced through the nucleolar organizer region of chromosome 2. These results indicate that the four cohesin proteins may form different complexes and/or perform additional functions during meiosis in plants, which are distinct from their essential function in sister chromatid cohesion.     

Probing the meiotic mechanism of intergenomic exchanges by genomic in situ hybridization on lampbrush chromosomes of unisexual <Emphasis Type="Italic">Ambystoma</Emphasis> (Amphibia: Caudata)

The meiotic mechanism of unisexual salamanders in the genus Ambystoma was previously explained by observing lampbrush chromosomes (LBCs). In polyploid unisexual females, a pre-meiotic endomitotic event doubles the chromosome number so that, after meiotic reduction, the mature eggs have the same ploidy as the female. It was assumed that synapses during meiotic I prophase, which result in observed bivalents, join duplicated sister chromosomes. Previous studies also found LBC quadrivalents in some oocytes that could be explained by occasional synapses between homologs. The discovery of widespread intergenomic exchanges among unisexual populations has prompted new speculations on this meiotic mechanism. Synapses that involve homeologous chromosomes may be frequent during meiosis and could be responsible for intergenomic exchanges and the high embryonic mortality of unisexuals. Furthermore, LBC quadrivalents may be established by associations between homeologous rather than homologous chromosomes. The present study investigated these two important aspects pertaining to the mechanism of intergenomic exchanges: the frequency of homeologous synapses and the relationship between homeologous associations and meiotic quadrivalents. We applied genomic in situ hybridization (GISH) on LBCs from oocytes of 14 triploid and two tetraploid unisexual females. Homeologous bivalents were not observed, and all 13 LBC quadrivalents that we found were the result of homologous synapses and were not associated with any homeologous or exchanged LBCs. Intergenomic exchanges were used as markers to compare the same chromosomes at meiotic diplotene and mitotic metaphase stages. We conclude that contemporary intergenomic exchanges are very rare, and no direct link exists between intergenomic exchanges and high embryonic mortality. The actual mechanisms and evolutionary implications of intergenomic exchanges appear to be complicated and difficult to assess. The application of GISH-type molecular cytogenetic techniques will help to improve our understanding of the role that intergenomic interactions play in the persistence of unisexual Ambystoma and other unisexual vertebrates.     

Differential association of SMC1α and SMC3 proteins with meiotic chromosomes in wild-type and SPO11-deficient male mice

SMC proteins are components of cohesin complexes that function in chromosome cohesion. We determined that SMC1α and SMC3 localized to wild-type mouse meiotic chromosomes, but with distinct differences in their patterns. Anti-SMC3 coincided with axial elements of the synaptonemal complex, while SMC1α was observed mainly in regions where homologues were synapsed. This pattern was especially visible in pachytene sex vesicles where SMC1α localized only weakly to the asynapsed regions. At diplotene, SMC3, but not SMC1α, remained bound along axial elements of desynapsed chromosomes. SMC1α and SMC3 were also found to localize along meiotic chromosome cores of Spo11 null spermatocytes, in which double-strand break formation required for DNA recombination and homologous pairing were disrupted. In Spo11−/− cells, SMC1α localization differed from SMC3 again, confirming that SMC1α is mainly associated with homologous or non-homologous synapsed regions, whereas SMC3 localized throughout the chromosomes. Our results suggest that the two cohesin proteins may not always be associated in a dimer and may function as separate complexes in mammalian meiosis, with SMC1α playing a more specific role in synapsis. In addition, our results indicate that cohesin cores can form independently of double-strand break formation and homologous pairing. This revised version was published online in July 2006 with corrections to the Cover Date.     

Localization of mouse Rad51 and Lim15 proteins on meiotic chromosomes at late stages of prophase 1

Background: In meiosis, eukaryotic chromosomes show a series of morphological changes, during which chromosomes synapse and recombine. To understand the mechanisms of the morphological changes and recombination of chromosomes, we examined stage-specific localization of the Rad51 and Lim15 proteins on the chromosomes in meiotic prophase 1. These proteins are homologous with the RecA protein and have general properties of searching and pairing of homologous DNA sequences. We used mouse chromosomes whose small sizes allow us to identify the locations of these proteins on the entire structures of the chromosomes.
Results: In the leptotene and zygotene stages, the Rad51 protein was present on chromatin loops of mouse testis chromosomes then the protein left the loops. In the pachytene stage, the Rad51 protein was present almost exclusively along the core of the synaptonemal complexes (SC). When the stage proceeded to diplotene, the protein was present in the synaptic regions of chromosomes, in particular, in the chiasma regions. The protein was not present on separated homologous SC cores. On the other hand, the Lim15 protein that was found on chromatin loops in early prophase 1, was present almost exclusively at both ends of the SC cores throughout the late stages of prophase 1.
Conclusion: The Rad51 and Lim15 proteins are present in chromatin loops when chromosomes form SC. The proteins may promote pairing of homologous DNA sequences that would lead formation of SC. The Rad51 protein in the SC cores may be involved in chiasma formation in late stages. The Lim15 protein, instead, may be involved in recombination in the telomeric region or in cohesion of sister chromatids for segregation.     

Cohesion by topology: sister chromatids interlocked by DNA

Sister chromatid cohesion is coupled with chromosome replication and influences chromosome segregation and intra-S repair. Specialized proteins, the cohesins, together with other pathways contribute to tether sister chromatids. In this issue of Genes & Development, Wang and colleagues (pp. 2426-2433 demonstrate that TopoIV, a type II DNA topoisomerase, modulates cohesion in Escherichia coli, by removing interlocked DNA junctions between sister chromatids. They propose that DNA precatenanes, arising during replication fork progression, hold sister chromatids together.     

Chromosome elimination in the interspecific hybrid medaka between <Emphasis Type="Italic">Oryzias latipes</Emphasis> and <Emphasis Type="Italic">O. hubbsi</Emphasis>

An interspecific hybrid medaka (rice fish) between Oryzias latipes and O. hubbsi is embryonically lethal. To gain an insight into the cellular and molecular mechanisms that cause the abnormalities occurring in the hybrid medaka, we investigated the behavior of chromosomes and the expression patterns of proteins responsible for the chromosome behavior. The number of chromosomes in the hybrid embryos gradually decreased to nearly half, since abnormal cell division with lagging chromosomes at anaphase eliminated the chromosomes from the cells. The chromosome lagging occurred at the first cleavage and continued throughout embryogenesis even after the midblastula transition. Fluorescent in-situ hybridization analyses revealed that the chromosomes derived from O. hubbsi are preferentially eliminated in both O. latipes–hubbsi and O. hubbsi–latipes embryos. Whole-mount immunocytochemical analyses using antibodies against α-tubulin, γ-tubulin, inner centromere protein, Cdc20, Mad2, phospho-histone H3 and cohesin subunits (SMC1α, SMC3 and Rad21) showed that the expression patterns of these proteins in the hybrid embryos are similar to those in the wild-type embryos, except for phospho-histone H3. Phospho-histone H3 present on chromosomes at metaphase was lost from normally separated chromosomes at anaphase, whereas it still existed on lagging chromosomes at anaphase, indicating that the lagging chromosomes remain in the metaphase state even when the cell has proceeded to the anaphase state. On the basis of these findings, we discuss the cellular and molecular mechanisms of chromosome elimination in the hybrid medaka.     

A Caenorhabditis elegans cohesion protein with functions in meiotic chromosome pairing and disjunction

We have studied four Caenorhabditis elegans homologs of the Rad21/Scc1/Rec8 sister-chromatid cohesion protein family. Based on the RNAi phenotype and protein localization, it is concluded that one of them, W02A2.6p, is the likely worm ortholog of yeast Rec8p. The depletion of C. elegans W02A2.6p (called REC-8) by RNAi, induced univalent formation and splitting of chromosomes into sister chromatids at diakinesis. Chromosome synapsis at pachytene was defective, but primary homology recognition seemed unaffected, as a closer-than-random association of homologous fluorescence in situ hybridization (FISH) signals at leptotene/zygotene was observed. Depletion of REC-8 also induced chromosome fragmentation at diakinesis. We interpret these fragments as products of unrepaired meiotic double-stranded DNA breaks (DSBs), because fragmentation was suppressed in a spo-11 background. Thus, REC-8 seems to be required for successful repair of DSBs. The occurrence of DSBs in REC-8-depleted meiocytes suggests that DSB formation does not depend on homologous synapsis. Anti-REC-8 immunostaining decorated synaptonemal complexes (SCs) at pachytene and chromosomal axes in bivalents and univalents at diakinesis. Between metaphase I and metaphase II, REC-8 is partially lost from the chromosomes. The partial loss of REC-8 from chromosomes between metaphase I and metaphase II suggests that worm REC-8 might function similarly to yeast Rec8p. The loss of yeast Rec8p from chromosome arms at meiosis I and centromeres at meiosis II coordinates the disjunction of homologs and sister chromatids at the two meiotic divisions.     

Dynamic molecular linkers of the genome: the first decade of SMC proteins

Structural maintenance of chromosomes (SMC) proteins are chromosomal ATPases, highly conserved from bacteria to humans, that play fundamental roles in many aspects of higher-order chromosome organization and dynamics. In eukaryotes, SMC1 and SMC3 act as the core of the cohesin complexes that mediate sister chromatid cohesion, whereas SMC2 and SMC4 function as the core of the condensin complexes that are essential for chromosome assembly and segregation. Another complex containing SMC5 and SMC6 is implicated in DNA repair and checkpoint responses. The SMC complexes form unique ring- or V-shaped structures with long coiled-coil arms, and function as ATP-modulated, dynamic molecular linkers of the genome. Recent studies shed new light on the mechanistic action of these SMC machines and also expanded the repertoire of their diverse cellular functions. Dissecting this class of chromosomal ATPases is likely to be central to our understanding of the structural basis of genome organization, stability, and evolution.     

Meiotic chromosome pairing in the normal human female

The synaptonemal complexes of oocytes from 16–22 week human fetuses were spread using detergent, and silver-stained for examination by light microscopy. Zygotene chromosome synapsis generally begins at the telomeres, without obvious prealignment, and proceeds towards the centromeres. Synapsis is not synchronous and longer bivalents may sometimes be completely paired before shorter ones. At pachytene, when pairing is usually complete, some regions presumed to correspond to the heterochromatic blocks of chromosomes 1, 9 and 16 may remain unpaired. Residual univalents are uncommon, and little interlocking is evident at this stage. Desynapsis indicating the beginning of diplotene frequently begins at the telomeres, although there is a general relaxation of pairing throughout the bivalents which become increasingly diffuse as diplotene proceeds. The total synaptonemal complex complement length at pachytene in the female is 519 μm, which is about twice that found in the human male. The implications of these results for genetic mapping are discussed.     

A model for chromosome structure during the mitotic and meiotic cell cycles

The chromosome scaffold model in which loops of chromatin are attached to a central, coiled chromosome core (scaffold) is the current paradigm for chromosome structure. Here we present a modified version of the chromosome scaffold model to describe chromosome structure and behavior through the mitotic and meiotic cell cycles. We suggest that a salient feature of chromosome structure is established during DNA replication when sister loops of DNA extend in opposite directions from replication sites on nuclear matrix strands. This orientation is maintained into prophase when the nuclear matrix strand is converted into two closely associated sister chromatid cores with sister DNA loops extending in opposite directions. We propose that chromatid cores are contractile and show, using a physical model, that contraction of cores during late prophase can result in coiled chromatids. Coiling accounts for the majority of chromosome shortening that is needed to separate sister chromatids within the confines of a cell. In early prophase I of meiosis, the orientation of sister DNA loops in opposite directions from axial elements assures that DNA loops interact preferentially with homologous DNA loops rather than with sister DNA loops. In this context, we propose a bar code model for homologous presynaptic chromosome alignment that involves weak paranemic interactions of homologous DNA loops. Opposite orientation of sister loops also suppresses crossing over between sister chromatids in favor of crossing over between homologous non-sister chromatids. After crossing over is completed in pachytene and the synaptonemal complex breaks down in early diplotene (= diffuse stage), new contractile cores are laid down along each chromatid. These chromatid cores are comparable to the chromatid cores in mitotic prophase chromosomes. As an aside, we propose that leptotene through early diplotene represent the missing G2 period of the premeiotic interphase. The new chromosome cores, along with sister chromatid cohesion, stabilize chiasmata. Contraction of cores in late diplotene causes chromosomes to coil in a configuration that encourages subsequent syntelic orientation of sister kinetochores and amphitelic orientation of homologous kinetochore pairs on the spindle at metaphase I.     

Rec8-containing cohesin maintains bivalents without turnover during the growing phase of mouse oocytes

During female meiosis, bivalent chromosomes are thought to be held together from birth until ovulation by sister chromatid cohesion mediated by cohesin complexes whose ring structure depends on kleisin subunits, either Rec8 or Scc1. Because cohesion is established at DNA replication in the embryo, its maintenance for such a long time may require cohesin turnover. To address whether Rec8- or Scc1-containing cohesin holds bivalents together and whether it turns over, we created mice whose kleisin subunits can be cleaved by TEV protease. We show by microinjection experiments and confocal live-cell imaging that Rec8 cleavage triggers chiasmata resolution during meiosis I and sister centromere disjunction during meiosis II, while Scc1 cleavage triggers sister chromatid disjunction in the first embryonic mitosis, demonstrating a dramatic transition from Rec8- to Scc1-containing cohesin at fertilization. Crucially, activation of an ectopic Rec8 transgene during the growing phase of Rec8(TEV)(/TEV) oocytes does not prevent TEV-mediated bivalent destruction, implying little or no cohesin turnover for ≥2 wk during oocyte growth. We suggest that the inability of oocytes to regenerate cohesion may contribute to age-related meiosis I errors.     

Holocentric chromosomes in meiosis. I. Restriction of the number of chiasmata in bivalents

The number of chiasmata in bivalents and the behaviour of chiasmata during the meiotic divisions were studied in Psylla foersteri (Psylloidea, Homoptera). Two chiasmata with a frequency of 97% and one or three chiasmata with frequencies of 2% and 0.9%, respectively, were observed in the largest bivalent in male meiosis. Meiosis was normal for the largest bivalents with one or two chiasmata, whereas bivalents with three chiasmata were not capable of completing anaphase I because of their inability to resolve the chiasma located in the middle. Consequently, the bivalent was seen as a laggard joining together two metaphase II daughter plates. Apparently, cells of this kind are eliminated. Inability to resolve the chiasma situated in the middle is attributed to the condensation process, which is unable to change the spatial orientation of successive chiasma loops in holocentric bivalents so that chiasma loops would be arranged perpendicular to each other at metaphase I. Thus they retain their parallel orientation from diplotene to metaphase I. Consequently, sister chromatid cohesion is exposed for release only in the outermost chiasmata but the chiasma in the middle continues to interlock the chromosomes in the bivalent. The elimination of the cells carrying bivalents with more than two chiasmata creates a strong selection against the formation of more than two chiasmata in holocentric bivalents.     

Localization of human SMC1 protein at kinetochores

Proper cohesion of sister chromatids is prerequisite for correct segregation of chromosomes during cell division. The cohesin multiprotein complex, conserved in eukaryotes, is required for sister chromatid cohesion. Human cohesin is composed of a stable heterodimer of the structural maintenance of chromosomes (SMC) family proteins, hSMC1 and hSMC3, and non-SMC components, hRAD21 and SA1 (or SA2). In yeast, cohesin associates with chromosomes from late G1 to metaphase and is required for the establishment and maintenance of both chromosome arm and centromeric cohesion. However, in human cells, the majority of cohesin dissociates from chromosomes before mitosis. Although it was recently shown that a small amount of hRAD21 localizes to the centromeres during metaphase, the presence of other cohesin components at the centromere has not been demonstrated in human cells. Here we report the mitosis-specific localization of hSMC1 to the kinetochores. hSMC1 is targeted to the kinetochore region during prophase concomitant with kinetochore assembly and remains through anaphase. Importantly, hSMC1 is targeted only to the active centromere on dicentric chromosomes. These results suggest that hSMC1 is an integral component of the functional kinetochore structure during mitosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Premature chromatid separation is not a useful diagnostic marker for Cornelia de Lange syndrome

Cornelia de Lange syndrome (CdLS) is a rare, multiple congenital anomaly/mental retardation syndrome characterized by clinical variability and caused by mutations in the NIPBL (50–60%), SMC1L1 and SMC3 genes (5%), which encode for proteins involved in sister chromatid cohesion. Almost all of the studies of premature chromatid separation (PCS) in CdLS patients have failed to demonstrate that it is specific to CdLS, thus making its diagnostic use controversial. In order to verify the diagnostic usefulness of PCS screening in CdLS, we analysed metaphase spreads from 29 CdLS patients and 24 controls using a rigorous protocol to induce PCS, and precise criteria to score the affected chromosomes. Following exclusion of significant intra-sample variation we scored under blind conditions 150 spreads from a single preparation of each case and computed the ratio between the number of prematurely separated chromatids and the total number of chromatids. The results indicate the extreme variability of PCS in both cohorts (CdLS: mean 2.8 ± 2.8%; controls: mean 4.0 ± 5.4%) and highlight the difficulty of PCS monitoring, especially when selecting the control population. The absence of any difference in the frequency of PCS between the patients and controls, or between patients with different clinical or genetic backgrounds, precludes its potential use as an additional diagnostic tool.