Regulation of histone acetylation during meiotic maturation in mouse oocytes

Histone acetylation is an important epigenetic modification implicated in the regulation of chromatin structure and, subsequently, gene expression. Global histone deacetylation was reported in mouse oocytes during meiosis but not mitosis. The regulation of this meiosis-specific deacetylation has not been elucidated. Here, we demonstrate that p34(cdc2) kinase activity and protein synthesis are responsible for the activation of histone deacetylases and the inhibition of histone acetyltransferases (HATs), respectively, resulting in deacetylation of histone H4 at lysine-12 (H4K12) during mouse oocyte meiosis. Temporal changes in the acetylation state of H4K12 were examined immunocytochemically during meiotic maturation using an antibody specific for acetylated H4K12. H4K12 was deacetylated during the first meiosis, temporarily acetylated around the time of the first polar body (PB1) extrusion, and then deacetylated again during the second meiosis. Because these changes coincided with the known oscillation pattern of p34(cdc2) kinase activity, we investigated the involvement of the kinase in H4K12 deacetylation. Roscovitine, an inhibitor of cyclin-dependent kinase activity, prevented H4K12 deacetylation during both the first and second meiosis, suggesting that p34(cdc2) kinase activity is required for deacetylation during meiosis. In addition, cycloheximide, a protein synthesis inhibitor, also prevented deacetylation. After PB1 extrusion, at which time H4K12 had been deacetylated, H4K12 was re-acetylated in the condensed chromosomes by treatment with cycloheximide but not with roscovitine. These results demonstrate that HATs are present but inactivated by newly synthesized protein(s) that is (are) not involved in p34(cdc2) kinase activity. Our results suggest that p34(cdc2) kinase activity induces the deacetylation of H4K12 and that the deacetylated state is maintained by newly synthesized protein(s) that inhibits HAT activity during meiosis.     

Changes in histone acetylation during mouse oocyte meiosis

We examined global changes in the acetylation of histones in mouse oocytes during meiosis. Immunocytochemistry with specific antibodies against various acetylated lysine residues on histones H3 and H4 showed that acetylation of all the lysines decreased to undetectable or negligible levels in the oocytes during meiosis, whereas most of these lysines were acetylated during mitosis in preimplantation embryos and somatic cells. When the somatic cell nuclei were transferred into enucleated oocytes, the acetylation of lysines decreased markedly. This type of deacetylation was inhibited by trichostatin A, a specific inhibitor of histone deacetylase (HDAC), thereby indicating that HDAC is able to deacetylate histones during meiosis but not during mitosis. Meiosis-specific deacetylation may be a consequence of the accessibility of HDAC1 to the chromosome, because HDAC1 colocalized with the chromosome during meiosis but not during mitosis. As histone acetylation is thought to play a role in propagating the gene expression pattern to the descendent generation during mitosis, and the gene expression pattern of differentiated oocytes is reprogrammed during meiosis to allow the initiation of a new program by totipotent zygotes of the next generation, our results suggest that the oocyte cytoplasm initializes a program of gene expression by deacetylating histones.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.     

流式细胞术检测组蛋白乙酰化水平方法的建立与应用

组蛋白去乙酰化酶抑制剂是近年来出现的一类新的抗肿瘤药物,在艾滋病等其他疾病中同样也受到关注。但是在基础和临床研究中,目前还缺乏统一可靠的组蛋白乙酰化水平的检测手段。本文利用全血和外周血单个核细胞,通过一系列的对比实验,比较了不同样品处理温度(冰上和室温)、破膜方法(细胞内因子染色破膜和核内因子染色破膜)、抗体剂量(抗体滴定)和抗体孵育时间(时间梯度)等实验条件对流式细胞术检测的影响,最终建立了一套基于流式细胞术的组蛋白乙酰化水平检测手段。同时,将优化后的流式细胞检测技术应用于西达本胺(目前国内唯一上市的组蛋白去乙酰化酶抑制剂)的体外实验和临床试验,结果均证明本文建立的组蛋白乙酰化流式细胞检测方法可以作为基础和临床研究中一个可靠、快速、便捷的检测手段。     

Meiotic competence and acetylation pattern of UV light classified mouse antral oocytes after meiotic arrest with isobutylmethylxanthine

Chromatin transformation from a diffused or NSN configuration to a compacted or SN shape that forms a ring around the nucleolus is regarded as one of the modifications necessary for successful embryonic development. But the process of the transformation is poorly understood. In this study we cultured mouse antral oocytes under meiotic arrest with IBMX for periods between 3 and 24 hr. We observed the chromatin status of the oocytes before and after culture under UV illumination. We reported here that the NSN configured oocytes transformed temporally through an intermediate form into the SN configuration while under meiotic arrest in vitro. Meiotic rate was improved in the NSN oocytes after the meiotic arrest but decreased in the SN oocytes. We also reported that chromatin of both the NSN and SN oocytes was acetylated and the two groups underwent the same pattern of H4/K5 deacetylation during meiotic maturation. We hypothesized that the transformation of mouse oocyte from the NSN to SN type may be time rather than oocyte size specific and the abrupt deacetylation of NSN oocyte during spontaneous maturation may explain its poor meiotic and developmental competence.     

Induction of meiotic maturation in mouse oocytes by adenosine analogs

In this study we have examined the meiosis-inducing influence of adenosine analogs in mouse oocytes. When a varied group of nucleosides and nucleotides were tested on overnight cultures of hypoxanthine-arrested, cumulus cell-enclosed oocytes (CEO), halogenated adenosine nucleosides, but not native adenosine, exhibited a significant meiosis-inducing capability. When tested under a variety of conditions, meiotic induction by 8-bromo-adenosine (8-Br-Ado) and a second adenosine analog, methylmercaptopurine riboside (MMPR), was especially potent in denuded oocytes (DO) compared to CEO and was not dependent on the type of inhibitor chosen to maintain meiotic arrest. Germinal vesicle breakdown (GVB) was stimulated with rapid kinetics and was preceded by an increase in AMP-activated protein kinase (AMPK) activity. Moreover, compound C, an inhibitor of AMPK, blocked the meiosis-inducing activities of both adenosine analogs. When tested for an effect on meiotic progression to metaphase II (MII) in spontaneously maturing CEO, 8-Br-Ado and the AMPK activator, 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), increased the percentage of MII-stage oocytes, but MMPR decreased this number. Adenosine and inhibitors of de novo purine synthesis had no effect on the completion of maturation, while compound C suppressed this process. These results support the proposition that oocyte AMPK mediates the positive influence of AICAR and 8-Br-Ado on both the initiation and completion of meiotic maturation. The role of AMPK in MMPR action is less clear.     

Effects of NO-synthase inhibitors on maturation mouse oocytes in cumulus-oocyte complexes

We studied the effects of various NO-synthase inhibitors on meiotic maturation of mouse oocytes in vitro in cumulus-oocyte complexes isolated from follicles of varying sizes. Selective and nonselective inhibitors of NO-synthase isoforms suppressed meiotic maturation of oocytes to varying degrees, which was expressed in a decreased number of oocytes at metaphase II. The results obtained suggest that the role of inducible form of NO-synthase (iNOS) increases with the development of follicles and oocytes.     

Analysis of aneuploidy rate in antral and ovulated mouse oocytes during female aging

Two forms of oocytes termed SN (surrounded nucleolus) and NSN (nonsurrounded nucleolus) differing for the spatial distribution of nuclear and nucleolar-associated chromatin have been described within the antral compartment of the ovary of a number of mammals. The biological significance of these two kind of oocytes is as yet not completely clear. In previous studies we have shown that prior to ovulation, mouse SN oocytes isolated from the antral compartment, matured and fertilized in vitro have a far better meiotic and developmental competence than NSN oocytes. Immediately after ovulation SN and NSN oocytes remaining in the antral compartment do not develop beyond the 2-cell stage. To further examine the correlation between chromatin distribution and meiotic competence of mouse antral oocytes, in the present study we have analyzed chromosome segregation at the first meiotic division in antral (SN and NSN) and in ovulated oocytes. SN and NSN oocytes were isolated before (48 h post PMSG injection) or after (15 h post–hCG injection) ovulation from ovaries of females of increasing age, they were cultured in vitro to metaphase II, and their aneuploidy rate was examined. Comparison of data obtained before and after ovulation highlights two main points: 1. Following ovulation a statistically significant increase of aneuploidy is observed in antral oocytes in most age groups and it is attributable to SN oocytes. 2. The aneuploidy rate of ovulated oocytes does not increase during female aging. We have found a correlation between chromatin distribution, hormonal status, and the incidence of aneuploidy during the oocyte first meiotic division. Mol. Reprod. Dev. 50 :305–312, 1998. © 1998 Wiley-Liss, Inc.     

Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes

The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system.     

X-inactivation is stably maintained in mouse embryos deficient for histone methyl transferase G9a

One of the two X chromosomes becomes inactivated during early development of female mammals. Recent studies demonstrate that the inactive X chromosome is rich in histone H3 methylated at Lys-9 and Lys-27, suggesting an important role for these modifications in X-inactivation. It has been shown that in the mouse Eed is required for maintenance of X-inactivation in the extraembryonic lineages. Interestingly, Eed associates with Ezh2 to form a complex possessing histone methyltransferase activity predominantly for H3 Lys-27. We previously showed that G9a is one of the histone methyltransferases specific for H3 Lys-9 and is essential for embryonic development. Here we examined X-inactivation in mouse embryos deficient for G9a. Expression of Xist, which is crucial for the initiation of X-inactivation, was properly regulated and the inactivated X chromosome was stably maintained even in the absence of G9a. These results demonstrate that G9a is not essential for X-inactivation.     

SPIN,a substrate in the MAP kinase pathway in mouse oocytes

The newly cloned gene Spin encodes a 30-kDa protein, a well-defined abundant molecule found in mouse oocytes and early embryos. This protein SPIN undergoes metaphase-specific phosphorylation and binds to the spindle. To understand the role of SPIN in oocyte meiosis, oocytes were treated with drugs that affect the cell cycle by activating or inactivating specific kinases. The posttranslational modification of SPIN in the treated oocytes was then investigated by one- and two-dimensional gel electrophoresis. Modification of SPIN is inhibited by treatment with 6-dimethylaminopurine (DMAP), suggesting that SPIN is phosphorylated by a serine-threonine kinase. Furthermore, SPIN from cycloheximide-treated oocytes that lack detectable MAP kinase activity is only partially phosphorylated, indicating that SPIN may be phosphorylated by the MOS/MAP kinase pathway. To confirm this observation, SPIN was analyzed in Mos-null mutant mice lacking MAP kinase activity. Normal posttranslational modification of SPIN did not occur in Mos-null mutant oocytes. In addition, there is reduced association of SPIN with the metaphase I spindle in Mos-null mutant oocytes, as determined by immunohistochemical analysis. These findings suggest that SPIN is a substrate in the MOS/MAP kinase pathway and further that this phosphorylation of SPIN may be essential for its interaction with the spindle. Mol. Reprod. Dev. 50:240–249, 1998. © 1998 Wiley-Liss, Inc.     

Role of N-terminus in function and dynamics of sirtuin 7: an in silico study

Abstract

The sirtuin family comprises seven NAD⁺-dependent histone deacetylases named SIRT1 to SIRT7. The least investigated SIRT7 is currently considered as a promising therapeutic target for cardiovascular diseases, diabetes and different types of cancer. So far, its structure was not experimentally resolved, except of a fragment of its N-terminus. The aim of this study was to create in silico model of SIRT7 containing its core together with N-terminus, which is known to affect the enzyme’s catalytic activity and to find pockets that could be targeted by structure-based virtual screening. Homology model of SIRT7 was prepared using X-ray structures of other sirtuins and a resolved fragment of the N-terminus of SIRT7 as templates. All atom-unbiased molecular dynamics simulations were performed. It was found that N-terminus of SIRT7 remains in spatial proximity of the catalytic core for considerable fraction of time, and therefore, it may affect its catalytic activity by helping the enzyme to hold the substrate peptide. It may also participate in holding and release of the cofactor. Preferred orientations of NAD⁺?and acetyl-lysine inside SIRT7 were found, with all components forming a stable complex. Molecular dynamics provided an ensemble of conformations that will be targeted with virtual screening. Reliable in silico structure of SIRT7 will be a useful tool in searching for its inhibitors, which can be potential drugs in cancer treatment.

Communicated by Ramaswamy H. Sarma     

MEK inhibitors block AICAR-induced maturation in mouse oocytes by a MAPK-independent mechanism

The present study was carried out to assess the possible role of mitogen-activated protein kinase (MAPK) in the meiosis-inducing action of the AMP-activated protein kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Cumulus cell-enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured 4 hr in Eagle's minimum essential medium containing dbcAMP plus increasing concentrations of AICAR or okadaic acid (OA). OA is a phosphatase inhibitor known to stimulate both meiotic maturation and MAPK activation and served as a positive control. Both OA and AICAR were potent inducers of meiotic resumption in mouse oocytes and brought about the phosphorylation (and thus, activation) of MAPK, but by different kinetics: MAPK phosphorylation preceded GVB in OA-treated oocytes, while that resulting from AICAR treatment appeared only after GVB. The MEK inhibitors, PD98059 and U0126, blocked the meiotic resumption induced by AICAR but not that induced by OA. Although the MEK inhibitors suppressed MAPK phosphorylation in both OA- and AICAR-treated oocytes, meiotic resumption was not causally linked to MAPK phosphorylation in either group. Furthermore, AICAR-induced meiotic resumption in Mos-null oocytes (which are unable to stimulate MAPK) was also abrogated by PD98059 treatment. A non-specific effect of the MEK inhibitors on AICAR accessibility to the oocyte was discounted by showing that they failed to suppress either nucleoside uptake or AICAR-stimulated phosphorylation of acetyl CoA carboxylase (ACC), a substrate of AMPK. The suppression of AICAR-induced maturation by MEK inhibitors must, therefore, be occurring by actions unrelated to MEK stimulation of MAPK; consequently, it would be prudent to consider this possible non-specific action of the inhibitors when they are used to block MAPK activation in mouse oocytes.     

Fer tyrosine kinase is required for germinal vesicle breakdown and meiosis-I in mouse oocytes

The control of microtubule and actin-mediated events that direct the physical arrangement and separation of chromosomes during meiosis is critical since failure to maintain chromosome organization can lead to germ cell aneuploidy. Our previous studies demonstrated a role for FYN tyrosine kinase in chromosome and spindle organization and in cortical polarity of the mature mammalian oocyte. In addition to Fyn, mammalian oocytes express the protein tyrosine kinase Fer at high levels relative to other tissues. The objective of the present study was to determine the function of this kinase in the oocyte. Feline encephalitis virus (FES)-related kinase (FER) protein was uniformly distributed in the ooplasm of small oocytes, but became concentrated in the germinal vesicle (GV) during oocyte growth. After germinal vesicle breakdown (GVBD), FER associated with the metaphase-I (MI) and metaphase-II (MII) spindles. Suppression of Fer expression by siRNA knockdown in GV stage oocytes did not prevent activation of cyclin dependent kinase 1 activity or chromosome condensation during in vitro maturation, but did arrest oocytes prior to GVBD or during MI. The resultant phenotype displayed condensed chromosomes trapped in the GV, or condensed chromosomes poorly arranged in a metaphase plate but with an underdeveloped spindle microtubule structure or chromosomes compacted into a tight sphere. The results demonstrate that FER kinase plays a critical role in oocyte meiotic spindle microtubule dynamics and may have an additional function in GVBD.     

Maturation‐associated Dbf4 expression is essential for mouse zygotic DNA replication

Cdc7 is an S‐phase‐promoting kinase (SPK) that is required for the activation of replication initiation complex assembly because it phosphorylates the MCM protein complex serving as the replicative helicase in eukaryotic organisms. Cdc7 activity is undetectable in immature mouse GV oocytes, although Cdc7 protein is already expressed at the same level as in mature oocytes or early one‐cell embryos at zygotic S‐phase, in which Cdc7 kinase activity is clearly detectable. Dbf4 is a regulatory subunit of Cdc7 and is required for Cdc7 kinase activity. Dbf4 is not readily detectable in immature GV oocytes but accumulates to a level similar to that in one‐cell embryos during oocyte maturation, suggesting that Cdc7 is already activated in unfertilized eggs (metaphase II). RNAi‐mediated knockdown of maternal Dbf4 expression prevents the maturation‐associated increase in Dbf4 protein, abolishes the activation of Cdc7, and leads to the failure of DNA replication in one‐cell embryos, demonstrating that Dbf4 expression is the key regulator of Cdc7 activity in mouse oocytes. Dormant Dbf4 mRNA in immature GV oocytes is recruited by cytoplasmic polyadenylation during oocyte maturation and is dependent on MPF activity via its cytoplasmic polyadenylation element (CPE) upstream of the hexanucleotide (HEX) in the 3′ untranslated region (3′UTR). Our results suggest that Cdc7 is inactivated in immature oocytes, preventing it from the unwanted phosphorylation of MCM proteins, and the oocyte is qualified by proper maturation to proceed following embryogenesis after fertilization through zygotic DNA replication.     

Stage‐dependent effects of epidermal growth factor on Ca2+ efflux in mouse oocytes

Epidermal growth factor (EGF) has received much attention recently for its positive effects on mammalian oocyte maturation and embryo development and its potential importance in cytoplasmic maturation of oocytes. Calcium (Ca²⁺) homeostasis in germinal vesicle stage oocytes has also been suggested to play a role in cytoplasmic maturation. This study examined the effects of EGF on Ca²⁺ mobilization as measured by its efflux from mouse oocytes at three time periods throughout maturation (0–4 hr, 4–8 hr, and 12 hr). Immature cumulus oocyte complexes (COCs) removed from the ovary for less than 4 hr exhibit oscillations in Ca²⁺ efflux that initiated 5–30 min following EGF stimulation. This response was not observed in COCs matured for 4–8 hr or 12 hr or in unstimulated 0–4 hr COCs. Denuded oocytes and cumulus cells did not show the same response to EGF (8.2 nM and 16.4 nM). Immunohistochemistry for detection of the EGF receptor along with EGF internalization studies showed that receptors are present both on cumulus cells and the oocyte but EGF appears to be internalized mainly by the cumulus cells. These data demonstrate that EGF induces oscillations in Ca²⁺ efflux in COCs 0–4 hr old and this response is mediated by the cumulus cells. Mol. Reprod. Dev. 53:244–253, 1999. © 1999 Wiley‐Liss, Inc.