Meiotic silencing and fragmentation of the male germline restricted chromosome in zebra finch

During male meiotic prophase in mammals, X and Y are in a largely unsynapsed configuration, which is thought to trigger meiotic sex chromosome inactivation (MSCI). In avian species, females are ZW, and males ZZ. Although Z and W in chicken oocytes show complete, largely heterologous synapsis, they too undergo MSCI, albeit only transiently. The W chromosome is already inactive in early meiotic prophase, and inactive chromatin marks may spread on to the Z upon synapsis. Mammalian MSCI is considered as a specialised form of the general meiotic silencing mechanism, named meiotic silencing of unsynapsed chromatin (MSUC). Herein, we studied the avian form of MSUC, by analysing the behaviour of the peculiar germline restricted chromosome (GRC) that is present as a single copy in zebra finch spermatocytes. In the female germline, this chromosome is present in two copies, which normally synapse and recombine. In contrast, during male meiosis, the single GRC is always eliminated. We found that the GRC in the male germline is silenced from early leptotene onwards, similar to the W chromosome in avian oocytes. The GRC remains largely unsynapsed throughout meiotic prophase I, although patches of SYCP1 staining indicate that part of the GRC may self-synapse. In addition, the GRC is largely devoid of meiotic double strand breaks. We observed a lack of the inner centromere protein INCENP on the GRC and elimination of the GRC following metaphase I. Subsequently, the GRC forms a micronucleus in which the DNA is fragmented. We conclude that in contrast to MSUC in mammals, meiotic silencing of this single chromosome in the avian germline occurs prior to, and independent of DNA double strand breaks and chromosome pairing, hence we have named this phenomenon meiotic silencing prior to synapsis (MSPS).     

Meiotic events at the centromeric heterochromatin: histone H3 phosphorylation, topoisomerase IIα localization and chromosome condensation

Mechanisms of chromosome condensation and segregation during the first meiotic division are not well understood. Resolution of recombination events to form chiasmata is important, for it is chiasmata that hold homologous chromosomes together for their oppositional orientation on the meiotic metaphase spindle, thus ensuring their accurate segregation during anaphase I. Events at the centromere are also important in bringing about proper attachment to the spindle apparatus. This study was designed to correlate the presence and activity of two proteins at the centromeric heterochromatin, topoisomerase II alpha (TOP2A) and histone H3, with the processes of chromosome condensation and individualization of chiasmate bivalents in murine spermatocytes. We tested the hypothesis that phosphorylation of histone H3 is a key event instigating localization of TOP2A to the centromeric heterochromatin and condensation of chromosomes as spermatocytes exit prophase and progress to metaphase. Activity of topoisomerase II is required for condensation of chromatin at the end of meiotic prophase. Histone H3 becomes phosphorylated at the end of prophase, beginning with its phosphorylation at the centromeric heterochromatin in the diplotene stage. However, it cannot be involved in localization of TOP2A, since TOP2A is localized to the centromeric heterochromatin throughout most of meiotic prophase. This observation suggests a meiotic function for TOP2A in addition to its role in chromatin condensation. The use of kinase inhibitors demonstrates that phosphorylation of histone H3 can be uncoupled from meiotic chromosome condensation; therefore other proteins, such as those constituting metaphase-promoting factor, must be involved. These results define the timing of important meiotic events at the centromeric heterochromatin and provide insight into mechanisms of chromosome condensation for meiotic metaphase.     

组蛋白H3Ser10磷酸化在家蚕精母细胞减数分裂中的动态分布

【目的】探究组蛋白H3Ser10磷酸化(H3Ser10ph)在家蚕Bombyx mori精母细胞减数分裂中的功能。【方法】解剖并分离家蚕4龄幼虫至蛹期精巢组织,通过丙烯酰胺凝胶包埋制备处于减数分裂不同时期的精巢组织玻片,以免疫荧光标记检测H3Ser10ph抗体在精母细胞减数分裂不同时期的定位特点。【结果】在家蚕有核精子精母细胞减数分裂过程中,组蛋白H3Ser10的磷酸化发生在粗线期染色体的特定位置,双线期H3Ser10ph信号逐渐减弱,至终变期时在染色体上完全检测不到磷酸化信号。随着细胞周期的进行,磷酸化信号又开始逐渐增强,减数第一次分裂中期时达到最高水平。当细胞进入减数第二次分裂前中期时,染色体臂上的H3Ser10ph信号消失,在靠近纺锤体微管的分裂面处有弥散的H3Ser10ph抗体的信号,减数第二次分裂末期,仅剩余非常微弱的H3Ser10ph信号残留于染色体的特定位置。在无核精子精母细胞减数分裂过程中,在中期I至末期I一直在染色体上有较均一的3Ser10ph信号,后期I时纺锤丝微管与赤道面平行。【结论】组蛋白H3Ser10磷酸化与家蚕有核精子和无核精子精母细胞减数分裂中染色质的动态变化相关。     

H3 Thr3 phosphorylation is crucial for meiotic resumption and anaphase onset in oocyte meiosis

Haspin-catalyzed histone H3 threonine 3 (Thr3) phosphorylation facilitates chromosomal passenger complex (CPC) docking at centromeres, regulating indirectly chromosome behavior during somatic mitosis. It is not fully known about the expression and function of H3 with phosphorylated Thr3 (H3T3-P) during meiosis in oocytes. In this study, we investigated the expression and sub-cellular distribution of H3T3-P, as well as its function in mouse oocytes during meiotic division. Western blot analysis revealed that H3T3-P expression was only detected after germinal vesicle breakdown (GVBD), and gradually increased to peak level at metaphase I (MI), but sharply decreased at metaphase II (MII). Immunofluorescence showed H3T3-P was only brightly labeled on chromosomes after GVBD, with relatively high concentration across the whole chromosome axis from pro-metaphase I (pro-MI) to MI. Specially, H3T3-P distribution was exclusively limited to the local space between sister centromeres at MII stage. Haspin inhibitor, 5-iodotubercidin (5-ITu), dose- and time-dependently blocked H3T3-P expression in mouse oocytes. H3T3-P inhibition delayed the resumption of meiosis (GVBD) and chromatin condensation. Moreover, the loss of H3T3-P speeded up the meiotic transition to MII of pro-MI oocytes in spite of the presence of non-aligned chromosomes, even reversed MI-arrest induced with Nocodazole. The inhibition of H3T3-P expression distinguishably damaged MAD1 recruitment on centromeres, which indicates the spindle assembly checkpoint was impaired in function, logically explaining the premature onset of anaphase I. Therefore, Haspin-catalyzed histone H3 phosphorylation is essential for chromatin condensation and the following timely transition from meiosis I to meiosis II in mouse oocytes during meiotic division.     

Persistence of histone H2AX phosphorylation after meiotic chromosome synapsis and abnormal centromere cohesion in poly (ADP-ribose) polymerase (Parp-1) null oocytes

In spite of the impact of aneuploidy on human health little is known concerning the molecular mechanisms involved in the formation of structural or numerical chromosome abnormalities during meiosis. Here, we provide novel evidence indicating that lack of PARP-1 function during oogenesis predisposes the female gamete to genome instability. During prophase I of meiosis, a high proportion of Parp-1^(−/−) mouse oocytes exhibit a spectrum of meiotic defects including incomplete homologous chromosome synapsis or persistent histone H2AX phosphorylation in fully synapsed chromosomes at the late pachytene stage. Moreover, the X chromosome bivalent is also prone to exhibit persistent double strand DNA breaks (DSBs). In striking contrast, such defects were not detected in mutant pachytene spermatocytes. In fully-grown wild type oocytes at the germinal vesicle stage, PARP-1 protein associates with nuclear speckles and upon meiotic resumption, undergoes a striking re-localization towards spindle poles as well as pericentric heterochromatin domains at the metaphase II stage. Notably, a high proportion of in vivo matured Parp-1^(−/−) oocytes show lack of recruitment of the kinetochore-associated protein BUB3 to centromeric domains and fail to maintain metaphase II arrest. Defects in chromatin modifications in the form of persistent histone H2AX phosphorylation during prophase I of meiosis and deficient sister chromatid cohesion during metaphase II predispose mutant oocytes to premature anaphase II onset upon removal from the oviductal environment. Our results indicate that PARP-1 plays a critical role in the maintenance of chromosome stability at key stages of meiosis in the female germ line. Moreover, in the metaphase II stage oocyte PARP-1 is required for the regulation of centromere structure and function through a mechanism that involves the recruitment of BUB3 protein to centromeric domains.     

HP1γ links histone methylation marks to meiotic synapsis in mice

During meiosis, specific histone modifications at pericentric heterochromatin (PCH), especially histone H3 tri- and dimethylation at lysine 9 (H3K9me3 and H3K9me2, respectively), are required for proper chromosome interactions. However, the molecular mechanism by which H3K9 methylation mediates the synapsis is not yet understood. We have generated a Cbx3-deficient mouse line and performed comparative analysis on Suv39h1/h2-, G9a- and Cbx3-deficient spermatocytes. This study revealed that H3K9me2 at PCH depended on Suv39h1/h2-mediated H3K9me3 and its recognition by the Cbx3 gene product HP1γ. We further found that centromere clustering and synapsis were commonly affected in G9a- and Cbx3-deficient spermatocytes. These genetic observations suggest that HP1γ/G9a-dependent PCH-mediated centromere clustering is an axis for proper chromosome interactions during meiotic prophase. We propose that the role of the HP1γ/G9a axis is to retain centromeric regions of unpaired homologous chromosomes in close alignment and facilitate progression of their pairing in early meiotic prophase. This study also reveals considerable plasticity in the interplay between different histone modifications and suggests that such stepwise and dynamic epigenetic modifications may play a pivotal role in meiosis.     

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

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

Histone H3 tails containing dimethylated lysine and adjacent phosphorylated serine modifications adopt a specific conformation during mitosis and meiosis

Condensation of chromatin, mediated in part by posttranslational modifications of histones, is essential for cell division during mitosis. Histone H3 tails are dimethylated on lysine (Kme2) and become phosphorylated on serine (Sp) residues during mitosis. We have explored the possibility that these double modifications are involved in the establishment of H3 tail conformations during the cell cycle. Here we describe a specific chromatin conformation occurring at Kme2 and adjacently phosphorylated S of H3 tails upon formation of a hydrogen bond. This conformation appears exclusively between early prophase and early anaphase of the mitosis, when chromatin condensation is highest. Moreover, we observed that the conformed H3Kme2Sp tail is present at the diplotene and metaphase stages in spermatocytes and oocytes. Our data together with results obtained by cryoelectron microscopy suggest that the conformation of Kme2Sp-modified H3 tails changes during mitosis and meiosis. This is supported by biostructural modeling of a modified histone H3 tail bound by an antibody, indicating that Kme2Sp-modified H3 tails can adopt at least two different conformations. Thus, the H3K9me2S10p and the H3K27me2S28p sites are involved in the acquisition of specific chromatin conformations during chromatin condensation for cell division.     

Behaviors of ATP‐dependent chromatin remodeling factors during maturation of bovine oocytes in vitro

The mammalian oocyte undergoes dynamic changes in chromatin structure to reach complete maturation. However, little known is about behaviors of ATP‐dependent chromatin remodeling factors (ACRFs) during meiosis. Here, we found that respective ACRFs may differently behave in the process of oocyte maturation in the bovine. All ACRFs interacted with oocytic chromatin at the germinal vesicle (GV) stage. Mi‐2 and hSNF2H disappeared from GV‐chromatin within 1 hr of in vitro culture whereas Brg‐1 and BAF‐170 were retained throughout germinal vesicle break down (GVBD). Brg‐1 was localized on the condensed chromatin outside, whereas BAF‐170 was entirely excluded from condensed chromatin. Thereafter, Brg‐1 and BAF‐170 interacted with metaphase I and metaphase II chromosomes. These results imply that Mi‐2 and hSNF2H may initiate the meiotic resumption, and Brg‐1 and BAF‐170 may support chromatin condensation during meiosis. In addition, DNA methylation and methylation of histone H3 at lysine 9 (H3K9) seem to be constantly retained in the oocyte chromatin throughout in vitro maturation. Inhibition of ACRF activity by treatment with the inhibitor apyrase led to retarded chromatin remodeling in bovine oocytes, thereby resulting in poor development of fertilized embryos. Therefore, these results indicate that precise behaviors of ACRFs during meiosis are critical for nuclear maturation and subsequent embryonic development in the bovine. Mol. Reprod. Dev. 77: 126–135, 2010. © 2009 Wiley‐Liss, Inc.     

Meiotic chromosome missegregation during apyrene meiosis in the gypsy moth,Lymantria dispar,is preceded by an aberrant prophase I

The gypsy moth, Lymantria dispar, produces two structurally and genetically distinct types of spermatozoa. The eupyrene spermatozoa are genetically haploid and structurally typical. The apyrene spermatozoa are anucleate and structurally different from eupyrene spermatozoa. To understand further the events contributing to meiotic chromosome missegregation in apyrene spermatocytes, we examined the progression of meiosis in these cells with respect to their eupyrene counterparts. Chromosomal bouquet formation and fusion of nucleolar organizing regions are disrupted in apyrene nuclei. In addition, the chromatin of apyrene nuclei is prematurely and extremely condensed compared with that of eupyrene nuclei. An antibody to the conserved synaptonemal complex protein 3 (SCP3) labeled eupyrene pachytene chromosomes, but not apyrene pachytene chromosomes. In addition, apyrene meiotic spindles are missing a subset of microtubules, which likely include kinetochore microtubules. Because the condensation behavior of meiotic chromatin in apyrene spermatocytes deviates from that of eupyrene spermatocytes, we examined the appearance and distribution of the phosphorylated form of histone H3, but no significant differences in histone H3 phosphorylation were found between apyrene and eupyrene spermatocytes. We argue that because a pachytene checkpoint is not initiated in apyrene spermatocytes, this system may provide a way to understand better the underlying biochemical connections between pairing, recombination, synapsis, kinetochore assembly and segregation of chromosomes during meiosis in a higher eukaryote.     

The centromeric heterochromatin of Costus spiralis: poorly methylated and transiently acetylated during meiosis

The centromeric region of Costus spiralis is characteristically composed of a small heterochromatic DAPI(+) band flanked by a discrete decondensed region. High concentrations of serine 10 of histone H3 (H3S10ph) around the DAPI(+) band in pachytene chromosomes and the location of this heterochromatin at the chromosome region directed towards the poles during metaphase-anaphase I confirm its integration into the centromeric region. Antibodies against both typical components of euchromatin histones (histone H4 acetylated at lysine 5 (H4K5ac) and histone H3 dimethylated at lysine 4 (H3K4me2)) and heterochromatin (dimethylated lysine 9 of H3 (H3K9me2) and anti-5-methylcytosine (5-mC)) were used to characterize the centromeric chromatin of this species during meiosis. In pachytene chromosomes, the decondensed terminal euchromatin of the chromosome arms were seen to be richer in H4K5ac and H3K4me2 histones, while the more condensed proximal region was relatively stronger labeled with anti-H3K9me2 and anti-5-methylcytosine (5-mC). The centromeric region itself, including the DAPI(+) band, was poor in all of these chromatin modifications, but it was highly enriched in H4K5ac at pachytene. Before and after this stage, the centromeric region was poorly labeled with anti-H4K5ac. Hypomethylation and hyperacetylation of any kind of heterochromatin has rarely been reported, and it may be related to the dominant role of the centromere domain over the heterochromatin repeats.     

Distribution and expression of phosphorylated histone H3 during porcine oocyte maturation

Phosphorylation modification of core histones is correlated well with diverse chromatin-based cell activities. However, its distribution pattern and primary roles during mammalian oocyte meiosis are still in dispute. In this study, by performing immunofluorescence and Western blotting, spatial distribution and temporal expression of phosphorylated serine 10 or 28 on histone H3 during porcine oocyte meiotic maturation were examined and distinct subcellular distribution patterns between them were presented. Low expression of phosphorylated H3/ser10 was detected in germinal vesicle. Importantly, following gradual dephosphorylation from germinal vesicle (GV) to late germinal vesicle (L-GV) stage, a transient phosphorylation at the periphery of condensed chromatin was re-established at early germinal vesicle breakdown (E-GVBD) stage, and then the dramatically increased signals covered whole chromosomes from pre-metaphase I (Pre-MI) to metaphase II (MII). Similarly, hypophosphorylation of serine 28 on histone H3 was also monitored from GV to E-GVBD, indicating dephosphorylation of histone H3 maybe involved in the regulation of meiotic resumption. Moreover, the rim staining on the chromosomes and high levels of H3/ser28 phosphorylation were observed in Pre-MI, MI, and MII stage oocytes. Based on above results, such stage-dependent dynamics of phosphorylation of H3/ser 10 and 28 may play specific roles during mammalian oocyte maturation.     

The Rad9 Gene of Coprinus Cinereus Encodes a Proline-Rich Protein Required for Meiotic Chromosome Condensation and Synapsis

The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stage of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus.