DNA,histone H1 and meiotin-1 immunostaining patterns along whole-mount preparations ofLilium longiflorum pachytene chromosomes

Meiotin-1 is a chromatin protein found in lily microsporocytes preceding and during meiosis. It reaches peak levels in the leptotene-pachytene interval. Here we report the pattern of immunostaining of meiotin-1 along pachytene chromosomes prepared using a whole-mount, synaptonemal complex spreading technique. Meiotin-1 immunostaining, like immunostaining for histone H1 and DNA, is found all along the lengths of the chromosomes. Unlike histone H1 and DNA immunostaining, there are several patches of particularly intense meiotin-1 immunostaining. The sites of intense staining may be clustered in thein vivo nucleus. The distribution of meiotin-1 along the width of the chromosomes was examined and compared with the pattern of histone H1 and DNA immunostaining. All three were distributed across the width of the chromosome, and did not appear to be preferentially associated with the synaptonemal complex. There was a slight suggestion that histone H1 and meiotin-1 may be located preferentially away from the synaptonemal complex. The temporal and spatial distribution of meiotin-1 in microsporocytes and tapetal cells, and the chemical and physical properties of meiotin-1, are discussed in the context of our hypothesis that meiotin-1 is a protein that functions to limit the degree of chromosome condensation during prophase I of meiosis.     

同型半胱氨酸对小鼠卵母细胞成熟过程中组蛋白表观遗传修饰的影响

目的 探讨卵母细胞发育过程中同型半胱氨酸（HCY）对组蛋白表观遗传修饰的影响。 方法 首先使用10只2周ICR雌性小鼠建立完整卵泡的体外培养体系,在卵母细胞发育早期,利用免疫组织化学方法,观察HCY对甲基化组蛋白H3K4、H3K9以及乙酰化组蛋白H3K9分布的影响;其次使用10只4周ICR雌性小鼠,利用卵母细胞的体外成熟培养体系,观察HCY对卵母细胞成熟过程的影响,同时利用实时定量PCR方法,观察在此成熟过程中HCY对卵母细胞内组蛋白乙酰化水平的调控酶GCN5和HDAC表达的影响。 结果 HCY明显抑制卵母细胞的体外成熟,在HCY作用下,甲基化组蛋白H3K4、H3K9以及乙酰化组蛋白H3K9的分布没有变化,但是表达强度降低,核呈现去浓缩的趋势。成熟过程中HCY并不改变基因GCN5的表达水平,却明显抑制卵母细胞内HDAC基因的表达。 结论 卵母细胞发育过程中高水平的同型半胱氨酸影响组蛋白的表观遗传修饰,HCY对卵母细胞核内组蛋白甲基化和乙酰化修饰的影响有可能是造成核染色体稳定性下降的重要原因。     

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.     

Chromatin condensation in terminally differentiating mouse erythroblasts does not involve special architectural proteins but depends on histone deacetylation

Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation and chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian erythroblast chromatin condensation, we used Friend virus-infected murine spleen erythroblasts that undergo terminal differentiation in vitro. Chromatin isolated from early and late-stage erythroblasts had similar levels of linker and core histones, only a slight difference in nucleosome repeats, and no significant accumulation of known developmentally regulated architectural chromatin proteins. However, histone H3(K9) dimethylation markedly increased while histone H4(K12) acetylation dramatically decreased and became segregated from the histone methylation as chromatin condensed. One histone deacetylase, HDAC5, was significantly upregulated during the terminal stages of Friend virus-infected erythroblast differentiation. Treatment with histone deacetylase inhibitor, trichostatin A, blocked both chromatin condensation and nuclear extrusion. Based on our data, we propose a model for a unique mechanism in which extensive histone deacetylation at pericentromeric heterochromatin mediates heterochromatin condensation in vertebrate erythroblasts that would otherwise be mediated by developmentally-regulated architectural proteins in nucleated blood cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Okadaic acid, an inhibitor of protein phosphatase 1 and 2A, induces premature separation of sister chromatids during meiosis I and aneuploidy in mouse oocytes in vitro

Recent advances in understanding some of the molecular aspects of chromosome segregation during mitosis and meiosis provide a background for investigating potential mechanisms of aneuploidy in mammalian germ cells. Numerous protein kinases and phosphatases have important functions during mitosis and meiosis. Alterations in these enzyme activities may upset the normal temporal sequence of biochemical reactions and cellular organelle modifications required for orderly chromosome segregation. Protein phosphatases 1 (PP1) and 2A (PP2A) play integral roles in regulating oocyte maturation (OM) and the metaphase–anaphase transitions. Mouse oocytes were transiently exposed invitro to different dosages (0, 0.01, 0.1, or 1.0 μg/ml) of the PP1 and PP2A phosphatase inhibitor okadaic acid (OA) during meiosis I and oocytes were cytogenetically analyzed. Significant (p < 0.05) OA dose-response increases in the frequencies of metaphase I (MI) arrested oocytes, MI oocytes with 80 chromatids instead of the normal 20 tetrads, and anaphase I–telophase I (AI–TI) oocytes with two groups of an unequal number of chromatids were found. Analysis of MII oocytes revealed significant (p < 0.05) increases in the frequencies of premature sister chromatid separation, single-unpaired chromatids, and hyperploidy. Besides showing that OA is aneugenic, these data suggest that OA-induced protein phosphatase inhibition upsets the normal kinase–phosphatase equilibrium during mouse OM, resulting in precocious removal of cohesion proteins from chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

A histone code in meiosis: the histone kinase, NHK-1, is required for proper chromosomal architecture in Drosophila oocytes

To promote faithful propagation of the genetic material during sexual reproduction, meiotic chromosomes undergo specialized morphological changes that ensure accurate segregation of homologous chromosomes. The molecular mechanisms that establish the meiotic chromosomal structures are largely unknown. We describe a mutation in a recently identified Histone H2A kinase, nhk-1, in Drosophila that leads to female sterility due to defects in the formation of the meiotic chromosomal structures. The metaphase I arrest and the karyosome, a critical prophase I chromosomal structure, require nucleosomal histone kinase-1 (NHK-1) function. The defects are a result of failure to disassemble the synaptonemal complex and to load condensin onto the mutant chromosomes. Embryos laid by nhk-1-/- mutant females arrest with aberrant polar bodies and mitotic spindles, revealing that mitosis is affected as well. We analyzed the role of Histone H2A phosphorylation with respect to the histone code hypothesis and found that it is required for acetylation of Histone H3 and Histone H4 in meiosis. These studies reveal a critical role for histone modifications in chromosome dynamics in meiosis and mitosis.     

The kinase haspin is required for mitotic histone H3 Thr 3 phosphorylation and normal metaphase chromosome alignment

Post-translational modifications of conserved N-terminal tail residues in histones regulate many aspects of chromosome activity. Thr 3 of histone H3 is highly conserved, but the significance of its phosphorylation is unclear, and the identity of the corresponding kinase unknown. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation of H3 Thr 3 in prophase and its dephosphorylation during anaphase. Furthermore we find that haspin, a member of a distinctive group of protein kinases present in diverse eukaryotes, phosphorylates H3 at Thr 3 in vitro. Importantly, depletion of haspin by RNA interference reveals that this kinase is required for H3 Thr 3 phosphorylation in mitotic cells. In addition to its chromosomal association, haspin is found at the centrosomes and spindle during mitosis. Haspin RNA interference causes misalignment of metaphase chromosomes, and overexpression delays progression through early mitosis. This work reveals a new kinase involved in composing the histone code and adds haspin to the select group of kinases that integrate regulation of chromosome and spindle function during mitosis and meiosis.     

Participation of EML6 in the regulation of oocyte meiotic progression in mice

The generation of a high-quality egg for reproduction requires faithful segregation of chromosome during oocyte meiosis. Here, we report that echinoderm microtubule-associated protein like 6 (EML6) is highly expressed in oocytes, and responsible for accurate segregation of homologous chromosomes in mice. Quantitative real-time RT-PCR and immunohistochemistry analyses revealed that EML6 was predominantly expressed by oocytes in the ovaries. Whole mount immunofluorescent staining showed that EML6 was colocalized with spindle microtubules in oocytes at various stages after meiotic resumption. This specialized localization was disrupted by nocodazole, the microtubule destabilizer, while enhanced by Taxol, a microtubule stabilizing reagent. In vivo knockdown of Eml6 expression by the specific siRNA resulted in chromosome misalignment and alteration in spindle dimension at both metaphase Ⅰ and Ⅱ stages, as well as the increased aneuploidy in the mature oocytes. Thus, these data suggest that EML family proteins participate in the control of oocyte meiotic division.     

HORMAD2 is essential for synapsis surveillance during meiotic prophase via the recruitment of ATR activity

Meiotic chromosome segregation requires homologous pairing, synapsis and crossover recombination during meiotic prophase. The checkpoint kinase ATR has been proposed to be involved in the quality surveillance of these processes, although the underlying mechanisms remain largely unknown. In our present study, we generated mice lacking HORMAD2, a protein that localizes to unsynapsed meiotic chromosomes. We show that this Hormad2 deficiency hampers the proper recruitment of ATR activity to unsynapsed chromosomes. Male Hormad2‐deficient mice are infertile due to spermatocyte loss as a result of characteristic impairment of sex body formation; an ATR‐ and γH2AX‐enriched repressive chromatin domain is formed, but is partially dissociated from the elongated sex chromosome axes. In contrast to males, Hormad2‐deficient females are fertile. However, our analysis of Hormad2/Spo11 double‐mutant females shows that the oocyte number is negatively correlated with the frequency of pseudo–sex body formation in a Hormad2 gene dosage‐dependent manner. This result suggests that the elimination of Spo11‐deficient asynaptic oocytes is associated with the HORMAD2‐dependent pseudo–sex body formation that is likely initiated by local concentration of ATR activity in the absence of double‐strand breaks. Our results thus show a HORMAD2‐dependent quality control mechanism that recognizes unsynapsis and recruits ATR activity during mammalian meiosis.     

The chromosomes ofFestuca pratensis Huds. (Poaceae): fluorochrome banding,heterochromatin and condensation

The karyotype of meadow fescueFestuca pratensis Huds. (2n=14) has been studied by means of fluorescence banding techniques. At-enriched heterochromatic bands were revealed in the metaphase chromosomes 1–6. A single GC-rich band was associated with the chromosome 2 secondary constriction. Delay of condensation of the heterochromatic regions was discovered. The heterochromatic bands reported are markers which allow identification of all meadow fescue chromosomes.     

Triploid origin of the gibel carp as revealed by 5S rDNA localization and chromosome painting

5S ribosomal DNA (rDNA) was isolated and sequenced from the gibel carp Carassius auratus gibelio with 162 chromosomes and crucian carp Carassius auratus with 100 chromosomes, and fluorescent probes for chromosome localization were prepared to ascertain the ploidy origin and evolutionary relationship between the two species. Using fluorescence in-situ hybridization (FISH), major 5S rDNA signals were localized to the short arms of three subtelocentric chromosomes in the gibel carp and to the short arms of two subtelocentrics in the crucian carp. In addition, some minor signals were detected on other chromosomes of both species. Simultaneously, six chromosomes were microdissected from the gibel carp metaphase spreads using glass needles, and the isolated chromosomes were amplified in vitro by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). Significantly, when the DOP-PCR-generated probes prepared from each single chromosome were hybridized, three same-sized chromosomes were painted in each gibel carp metaphase, whereas only two painted chromosomes were observed in each crucian carp metaphase spread. The data indicate that gibel carp is of triploid origin in comparison with diploid crucian carp.     

Meiotin-1, a Meiosis-enriched Protein Present in Normal Leptotene Chromosomes and Lacking in Precociously Condensed Leptotene Chromosomes

During mitotic prophase, chromosomes progressively compact to their metaphase length. In contrast, meiotic chromosomes condense moderately until late in prophase I, then they condense more dramatically (coil) to their fully condensed state. Meiotin-1 is a meiosis-enriched, chromosomal protein. We propose that it delays coiling until after reciprocal genetic exchange. We have used immunoblotting and immunocytochemistry with normal lily cells undergoing meiosis to demonstrate that meiotin-1 is present during the early portions of prophase I, but diminishes at the time when meiotic chromosomes begin to coil. Additionally, we have examined lily meiotic nuclei undergoing the reversible phenomenon of precocious leptotene chromosome condensation (precocious coiling). The leptotene chromosomes that are precociously condensed lack meiotin-1 immunostaining. Furthermore nuclei returning to the normal state of moderate prophase I condensation acquire meiotin-1.     

Turning it on and off: M-phase promoting factor during meiotic maturation and fertilization

Maturation (M-phase) promoting factor (MPF) plays a pivotal role in oocytes during their maturation. This review concentrates on its function at three important time-points. First, its activation during meiotic progression from prophase I arrest at germinal vesicle breakdown. Second, its role during the transition from meiosis I to meiosis II, a defining feature of meiosis involving segregation of homologous chromosomes. Third, maintenance of its activity at metaphase II arrest and the necessity for its destruction during oocyte activation. An understanding of how oocytes switch it on and turn it off underpins much of the basic cell biology of oocyte maturation.     

Meiotic behavior of the X1X2Y1Y2 quadrivalent of the primateAlouatta caraya

A multiple sex chromosome system was found in three unrelated individuals of the primateAlouatta caraya. This mechanism is originated by a translocation between the Y chromosome and one of the autosomes (A7). Mitotic karyotypes show two small, acrocentric chromosomes (A^Y and Y^A), which are the translocation products. In metaphase I of male meiosis, there is a very long chain quadrivalent in which the order of the element is: X–Y^A–A7–A^Y. Segregation in the quadrivalent is alternate and gives balanced products. Synaptonemal complex karyotypes at pachytene show the structure of the quadrivalent made by the four axes. There is a slight difference in the relative length of A^Y and Y^A and the kinetochore of A7 aligns with that of A^Y. The synaptic pattern and changes in the quadrivalent during pachytene are described. Thin sections of the quadrivalent body show that the chromatin packing in the sex chromosome region is different from that of the autosomal region. This X1X2Y1Y2/X1X1XX2 sex chromosome system may be extended among other members of the genusAlouatta.accepted for publication by M. Schmid     

G-banding and chromosome condensation in the ant,Tapinoma nigerrimum

Well-defined G-bands were obtained on metaphase chromosomes fromTapinoma nigerrimum using trypsin and warm 2×SSC in sequence. The G-banded pattern allowed the identification of all chromosomes. Evidence for asynchronous condensation of the chromosomes of this species is provided. Different banding patterns were obtained when metaphase chromosomes were stained with DA/DAPI alone and with DA/DAPI after a standard G-banding procedure. The G-banding phenomenon is discussed using the result obtained.accepted for publication by H. C. Macgregor     

Assembly of the SMRT-histone deacetylase 3 repression complex requires the TCP-1 ring complex

The acetylation of histone tails is a primary determinant of gene activity. Histone deacetylase 3 (HDAC3) requires the nuclear receptor corepressor SMRT for HDAC enzyme activity. Here we report that HDAC3 interacts with SMRT only after priming by cellular chaperones including the TCP-1 ring complex (TRiC), which is required for proper folding of HDAC3 in an ATP-dependent process. SMRT displaces TRiC from HDAC3, yielding an active HDAC enzyme. The SMRT-HDAC3 repression complex thus joins the VHL-elongin BC tumor suppression complex and the cyclin E-Cdk2 cell cycle regulation complex as critical cellular machines requiring TRiC for proper assembly and function. The strict control of HDAC3 activity underscores the cellular imperative that histone deacetylation occur only in targeted regions of the genome.