HDAC3 inhibition disrupts the assembly of meiotic apparatus during porcine oocyte maturation

Histone deacetylases (HDACs) are involved in a wide array of biological processes. However, the role of HDAC3 in porcine oocytes remains unclear. In the current study, we examine the effects of HDAC3 inhibition on porcine oocyte maturation using RGFP966, a selective HDAC3 inhibitor. We find that suppression of HDAC3 activity prevents not only the expansion of cumulus cells but also the meiotic progression of oocytes. It is interesting to note that HDAC3 displays a spindle-like distribution pattern as the porcine oocytes enter meiosis. In line with this, confocal microscopy reveals the high frequency of spindle defects and chromosomal congression failure in metaphase oocytes exposed to RGFP966. Moreover, HDAC3 inhibition results in the hyperacetylation of α-tubulin during oocyte meiosis. These findings indicate that HDAC3 activity might control the microtubule stability via the deacetylation of tubulin, which is critical for maintaining the proper spindle assembly, accurate chromosome separation, and orderly meiotic progression during porcine oocyte maturation.     

The effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

In order to study the effects of ubiquitin-proteasome pathway (UPP) on mouse oocyte meiosis and cleavage, oocytes undergoing maturation and parthenogenetic activation and 1-cell embryos were treated with lactacystin, a specific inhibitor of proteasome. The results indicated that the rate of GVBD was not influenced by the treatment, but polar body extrusion, parthenogenesis and first cleavage were inhibited. Immunofluorescent staining using anti β-tubulin antibody indicated that the continuous treatment of lactacystin from GV stage disorganized microtubules and spindle assembly. When metaphase stage oocytes were treated with the drug, the already formed spindle structure was not affected, but the oocytes were arrested at metaphases. The 1-cell embryos were arrested at interphase or metaphase of first mitosis when they were incubated in the drug. Proteasome regulatory subunit PA700 was located in the spindle region, as indicated by immunofluorescence. These results suggest that UPP has effects on the process of oocyte meiosis and early cleavage in many aspects, including normal organization of spindle at prophase and segregation of chromosomes at anaphase for normal meiosis.     

The effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

Many studies have shown that the ubiq-uitin-proteasome pathway (UPP) for the degradation of short-lived proteins plays a key role in regulating cell cycle progression[1—3]. At least two distinct prote-olytic pathways are required for cell cycle process. The first pathway promotes transition from G1 to S phase, and the second initiates the onset of anaphase and exit from mitosis. The inhibition of UPP will re-sult in the blockage of cell cycle process. The knowl-edge of the role of UPP in…     

PP2A regulates kinetochore-microtubule attachment during meiosis I in oocyte

Studies using in vitro cultured oocytes have indicated that the protein phosphatase 2A (PP2A), a major serine/threonine protein phosphatase, participates in multiple steps of meiosis. Details of oocyte maturation regulation by PP2A remain unclear and an in vivo model can provide more convincing information. Here, we inactivated PP2A by mutating genes encoding for its catalytic subunits (PP2Acs) in mouse oocytes. We found that eliminating both PP2Acs caused female infertility. Oocytes lacking PP2Acs failed to complete 1^st meiotic division due to chromosome misalignment and abnormal spindle assembly. In mitosis, PP2A counteracts Aurora kinase B/C (AurkB/C) to facilitate correct kinetochore-microtubule (KT-MT) attachment. In meiosis I in oocyte, we found that PP2Ac deficiency destabilized KT-MT attachments. Chemical inhibition of AurkB/C in PP2Ac-null oocytes partly restored the formation of lateral/merotelic KT-MT attachments but not correct KT-MT attachments. Taken together, our findings demonstrate that PP2Acs are essential for chromosome alignments and regulate the formation of correct KT-MT attachments in meiosis I in oocytes.     

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.     

CDC6 regulates both G2/M transition and metaphase-to-anaphase transition during the first meiosis of mouse oocytes

Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro-metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.     

Overexpression of cyclin A1 promotes meiotic resumption but induces premature chromosome separation in mouse oocyte

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

The regulation of competence to replicate in meiosis by Cdc6 is conserved during evolution

DNA replication licensing is an important step in the cell cycle at which cells become competent for DNA replication. When the cell cycle is arrested for long periods of time, this competence is lost. This is the case for somatic cells arrested in G0 or vertebrate oocytes arrested in G2. CDC6 is a factor involved in replication initiation competence which is necessary for the recruitment of the MCM helicase complex to DNA replication origins. In Xenopus, we have previously shown that CDC6 is the only missing replication factor in the oocyte whose translation during meiotic maturation is necessary and sufficient to confer DNA replication competence to the egg before fertilization (Lemaitre et al., 2002: Mol Biol Cell 13:435-444; Whitmire et al., 2002: Nature 419:722-725). Here, we report that this oogenesis control has been acquired by metazoans during evolution and conserved up to mammals. We also show that, contrary to eukaryotic metazoans, in S. pombe cdc18 (the S. pombe CDC6 homologue), CDC6 protein synthesis is down regulated during meiosis. As such, the lack of cdc18 prevents DNA replication from occurring in spores, whereas the presence of cdc6 makes eggs competent for DNA replication.     

Pharmacological inhibition of HDAC6 attenuates endothelial barrier dysfunction induced by thrombin

Background

Endothelial barrier dysfunction (EBD) involves microtubule disassembly and enhanced cell contractility. Histone deacetylase 6 (HDAC6) deacetylates α-tubulin, and thereby destabilizes microtubules. This study investigates a role for HDAC6 in EBD.

Methods

EBD was induced with thrombin ± HDAC6 inhibitors (tubacin and MC1575), and assessed by transendothelial electrical resistance (TEER). Markers for microtubule disassembly (α-tubulin and acetylated α-tubulin) and contraction (phosphorylated myosin light chain 2, P-MLC2) were measured using immunoblots and immunofluorescence.

Results and conclusion

Thrombin induced a ∼50% decrease in TEER that was abrogated by the HDAC6 inhibitors. Moreover, inhibition of HDAC6 diminished edema in the lung injured by lipopolysaccharide. Lastly, inhibition of HDAC6 attenuated thrombin-induced microtubule disassembly and P-MLC2. Our results suggest that HDAC6 can be targeted to limit EBD.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.     

Microtubule-associated deacetylase HDAC6 promotes angiogenesis by regulating cell migration in an EB1-dependent manner

Angiogenesis, a process by which the preexisting blood vasculature gives rise to new capillary vessels, is associated with a variety of physiologic and pathologic conditions. However, the molecular mechanism underlying this important process remains poorly understood. Here we show that histone deacetylase 6 (HDAC6), a microtubule-associated enzyme critical for cell motility, contributes to angiogenesis by regulating the polarization and migration of vascular endothelial cells. Inhibition of HDAC6 activity impairs the formation of new blood vessels in chick embryos and in angioreactors implanted in mice. The requirement for HDAC6 in angiogenesis is corroborated in vitro by analysis of endothelial tube formation and capillary sprouting. Our data further show that HDAC6 stimulates membrane ruffling at the leading edge to promote cell polarization. In addition, microtubule end binding protein 1 (EB1) is important for HDAC6 to exert its activity towards the migration of endothelial cells and generation of capillary-like structures. These results thus identify HDAC6 as a novel player in the angiogenic process and offer novel insights into the molecular mechanism governing endothelial cell migration and angiogenesis.     

Phosphorylation of histone H2A is associated with centromere function and maintenance in meiosis

Histone phosphorylation is dynamically regulated during cell division, for example phosphorylation of histone H3 (H3)-Ser10, H3-Thr11 and H3-Ser28. Here we analyzed maize (Zea mays L) for Thr133-phosphorylated histone H2A, which is important for spindle checkpoint control and localization of the centromere cohesion protector Shugoshin in mammals and yeast. Immunostaining results indicate that phosphorylated H2A-Thr133 signals bridged those of the centromeric H3 histone variant CENH3 by using a plant displaying yellow fluorescent protein-CENH3 signals and H2A-Thr133 is phosphorylated in different cell types. During mitosis, H2A-Thr133 phosphorylation becomes strong in metaphase and is specific to centromere regions but drops during later anaphase and telophase. Immunostaining for several maize dicentric chromosomes revealed that the inactive centromeres have lost phosphorylation of H2A-Thr133. During meiosis in maize meiocytes, H2A phosphorylation becomes strong in the early pachytene stage and increases to a maximum at metaphase I. In the maize meiotic mutant afd1 (absence of first division), sister chromatids show equational separation at metaphase I, but there are no changes in H2A-Thr-133 phosphorylation during meiosis compared with the wild type. In sgo1 mutants, sister chromatids segregate randomly during meiosis II, and phosphorylation of H2A-Thr-133 is observed on the centromere regions during meiosis II. The availability of such mutants in maize that lack sister cohesion and Shugoshin indicate that the signals for phosphorylation are not dependent on cohesion but on centromere activity.     

Effects of aromatase inhibition on in vitro follicle and oocyte development analyzed by early preantral mouse follicle culture

In vivo studies on folliculogenesis have documented a relation among intrafollicular steroid content, follicle growth, and oocyte development. This study examined how profound changes in androgen/estrogen ratio would affect mouse in vitro follicular development. Arimidex, a potent follicular aromatase inhibitor was used for this purpose. Early preantral follicles were cultured for 12 days up to the preovulatory stage. Oocyte's meiotic maturation, spindle and chromosome configurations, in vitro fertilization and preimplantation embryo development were evaluated. Compared to controls, Arimidex reduced E2 concentration in follicle culture medium by a factor 1000, and an expected simultaneous accumulation of testosterone was measured in the conditioned medium. Arimidex treatment provoked a dose-dependent earlier differentiation of the granulosa cells as judged by an earlier antrallike cavity formation and slightly elevated basal progesterone secretion. Follicle survival exceeded 98% in all groups and all follicles responded normally to HCG/EGF addition on day 12 by cumulus mucification. By the HCG ovulatory challenge, progesterone output was reduced in Arimidex supplemented groups suggesting preovulatory luteinization. These results indicate that in vitro mouse follicles can develop normally under very low levels of estrogens and that a local androgen increase by a factor 3 is not atretogenic. Oocyte growth did not differ among culture conditions. Arimidex treatment induced a dose dependent enhancement of GVBD and polar body formation rate in response to HCG at the end of culture. Spindle and chromosome analyses demonstrated that in all groups, 90% of the oocytes which extruded a polar body had also reached the MII stage. While most of the cultured MII oocytes had a normal spindle and well aligned chromosomes, significantly less oocytes were fertilized in the groups cultured in the presence of Arimidex. Once fertilized, however, there was found to be no difference for preimplantation embryo development between controls and Arimidex treatment. These data suggest that in mice a pronounced estrogenic environment is not essential for in vitro folliculogenesis. Drastic changes in the intrafollicular steroid concentrations do not disrupt meiotic maturation nor compromise early preimplantation development, but adversely affect fertilization of in vitro grown oocytes.