Aging alters histone H3 lysine 4 methylation in mouse germinal vesicle stage oocytes.

Decreasing oocyte competence with maternal aging is a major factor in mammalian infertility. One of the factors contributing to this infertility is changes to chromatin modifications, such as histone acetylation in old MII stage oocytes. Recent studies indicate that changes in histone acetylation at MII arise at the germinal vesicle (GV) stage. We hypothesised that histone methylation could also change in old GV oocytes. To test this hypothesis, we examined mono-, di- and trimethylation of histone H3 lysine 4 (H3K4 me1, me2 and me3, respectively) in young and older oocytes from 6-8- and 42-44-week-old mice, respectively. We found that H3K4 me2 and me3 decreased in older compared with young GV oocytes (100% vs. 81% and 100% vs. 87%, respectively; P<0.05). H3K4 me2 later increased in older MII oocytes (21% vs. 56%; P<0.05). We also examined the expression of genes encoding the H3K4 demethylases lysine (K)-specific demethylase 1A (Kdm1a) and retinol binding protein 2 (Rbp2). Expression of Kdm1a increased at both the mRNA and protein levels in older GV oocytes, but decreased in older MII oocytes (P<0.05), and was negatively correlated with H3K4 me2 levels. Conversely, expression of Rbp2 mRNA and protein decreased in older GV oocytes (P<0.05), and this was not correlated with H3K4 me3 levels. Finally, we showed that inhibition of Kdm1a of older oocytes at the GV stage restored levels of H3K4 me2 at the MII stage to those seen in 'young' oocytes (41% vs. 38%; P>0.05). These results suggest that changes in expression of H3K4 me2 and Kdm1a in older GV oocytes may represent a molecular mechanism underlying human infertility caused by aging.

Dynamic patterns of histone H3 lysine 4 methyltransferases and demethylases during mouse preimplantation development.

Extensive and dynamic chromatin remodeling occurs after fertilization, including DNA methylation and histone modifications. These changes underlie the transition from gametic to embryonic chromatin and are thought to facilitate early embryonic development. Histone H3 lysine 4 methylation (H3K4me) is an important epigenetic mechanism that associates with gene-specific activation and functions in development. However, dynamic regulation of H3K4me during early embryonic development remains unclear. Herein, the authors examined the dynamic changes of H3K4me and its key regulators (Ash1l, Ash2l, Kmt2a, Kmt2b, Kmt2c, Setd1a, Setd7, Kdm1a, Kdm1b, Kdm5a, Kdm5b, Kdm5c, and Kdm5d) in mouse oocytes and preimplantation embryos. An increase in levels of H3K4me2 and me3 was observed at the one- to two-cell stages (P < 0.05), corresponding to the period of embryonic genome activation (EGA). Subsequently, the H3K4me2 level dramatically decreased at the four-cell stage and remained at low level until the blastocyst stage (P < 0.05), whereas the H3K4me3 level transiently decreased in the four-cell embryos but steadily increased to the peak in the blastocysts (P < 0.05). The high level of H3K4me2 during the EGA was coinciding with a peak expression of its methyltransferase, ASH2L, which may stabilize this methylation level during this period. Correspondingly, a concomitant decrease in levels of its demethylases, KDM5B and KDM1A, was observed. H3K4me3 was correlated to the expression of its methyltransferase (KMT2B) and demethylase (KDM5A). Thus, these enzymes may function for the EGA and the first lineage segregation in preimplantation mouse embryos.

Effects of hyperoxia on transdifferentiation of primary cultured typeII alveolar epithelial cells from premature rats.

Hyperoxia exposure is a significant risk factor for the impaired alveolarization characteristic of bronchopulmonary dysplasia. Type II alveolar epithelial cells (AECIIs) may serve as "alveolar stem cells" to transdifferentiate into type I alveolar epithelial cells (AECIs). Here, we show that hyperoxia is capable of inducing transdifferentiation of AECIIs in premature rats in vitro. Hyperoxia-induced transdifferentiation was characterized by typical morphological changes, inhibition of cellular proliferation, decline in expression rate of Ki67, accumulation of cells in the G(1) phase of the cell cycle, increased expression of AECI-specific protein aquaporin 5, and decreased expression of AECII-associated protein surfactant protein C. These results suggest that hyperoxia may induce transdifferentiation of AECIIs into AECIs and the transdifferentiation may be responsible for repairing early lung injury.

[Histone demethylase LSD1 and its biological functions].

Discovery of histone lysine specific demethylase 1 (LSD1) indicates that even histone methylation is reversible. Structural analysis shows that LSD1 is a flavin-dependent amine oxidase, which is able to catalyze the specific removal of methyl groups from mono- and dimethylated Lys4 and Lys9 of histone H3. Functional studies demonstrate that LSD1 regulates activation and inhibition of gene transcription in the nucleus, which is known as the innermost gene switch of cells. LSD1 plays important roles in embryonic development and tumorigenesis. Here, we review recent insights into the structure and chemical mechanism of LSD1, and its regulatory roles in development and cancer.

Effect of nitric oxide on the proliferation of AGS gastric cancer cells.

BACKGROUND AND OBJECTIVE: Nitric oxide (NO) is involved in many physiologic and pathologic processes. As an important biologic mediator, NO has been the focus of cancer study for its function in tumorigenesis, tumor progression, and death. This study investigated the effect of NO donor sodium nitroprusside (SNP) on the growth and proliferation of gastric cancer cell line AGS. METHODS: The growth inhibition of AGS cells was analyzed using MTT assay. The cell cycle was measured using flow cytometry. The changes of mRNA expression of proliferating cell nuclear antigen (PCNA) and caspase-3 were examined using reverse transcriptase polymerase chain reaction (RT-PCR), and the protein expressions of PCNA and caspase-3 were analyzed using Western blot. RESULTS: Dose-dependent SNP inhibited cell growth and proliferation. When the AGS cells were treated with SNP at 100, 500, 1000, 1500, and 2000 mumol/L for 24 h, the growth inhibition rates were (2.02 +/- 2.96)%, (10.82 +/- 2.21)%, (18.95 +/- 3.35)%, (26.88 +/- 2.54)%, and (42.57 +/- 1.27)%, respectively (P < 0.05). SNP altered the cell cycle in AGS cells. Compared with the control group, treatment with SNP at 100, 500, 1000, 1500, and 2000 mumol/L for 24 h reduced the number of cells in the S phase by 2.29%, 7.8%, 11.34%, 20.49%, and 23.6%, respectively, and enhanced the number of cells in the G1/G0 phases by 3.33%, 9.3%, 13.46%, 21.37%, and 24.73%, respectively (P < 0.05). With the increasing concentration and action time of SNP, the expressions of PCNA mRNA and protein decreased. The expression of caspase-3 mRNA remained unchanged, but procaspase-3 was activated. CONCLUSION: NO not only inhibits cell growth and proliferation, but also induces apoptosis in gastric cancer cells, and such effects of NO showed significant dose-dependent activity.

Inheritance of histone H3 methylation in reprogramming of somatic nuclei following nuclear transfer.

Successful cloning requires reprogramming of epigenetic information of the somatic nucleus to an embryonic state. However, the molecular mechanisms regarding epigenetic reprogramming of the somatic chromatin are unclear. Herein, we transferred NIH3T3 cell nuclei into enucleated mouse oocytes and evaluated the histone H3 dimethyl-lysine 4 (H3K4me2) dynamics by immunocytochemistry. A low level of H3K4me2 in the somatic chromatin was maintained in pseudo-pronuclei. Unlike in vitro fertilized (IVF) embryos, the methylation level of nuclear transfer (NT) embryos was significantly increased at the 8-cell stage. NT embryos showed lower H3K4me2 intensity than IVF embryos at the 2-cell stage, which is when the mouse embryonic genome is activated. Moreover, the H3K4me2 signal was weak in the recloned embryos derived from single blastomeres of the NT embryos, whereas it was intense in those from IVF embryos. Two imprinted genes, U2afbp-rs and Xist, were abnormally transcribed in cloned embryos compared with IVF embryos, and this was partly correlated to the H3K4me2 level. Our results suggest that abnormal reprogramming of epigenetic markers such as histone acetylation and methylation may lead to dysregualtion of gene expression in cloned embryos.

Role of histone methylation in zygotic genome activation in the preimplantation mouse embryo.

Numerous previous studies demonstrated that gene expression was influenced by histone modifications. However, little information is available about the relation of histone methylation with embryonic gene expression. Here, we examine the significance of histone H3 dimethyl-lysine 4 (H3K4me2) during mouse zygotic genome activation (ZGA) by inhibiting demethylation with the specific histone H3 lysine 4 demethylase inhibitor bisguanidine 1c (1c). A 1c treatment of one-cell embryos did not significantly affect the level of eIF-4C transcripts but did affect Oct4 levels by the two-cell stage. Furthermore, 1c treatment significantly inhibited cleavage of the embryos to the four-cell stage (from 82.7% to 18.2%), and the inhibitory effect was identified to be irreversible. These results suggest that histone methylation may be closely correlated with the formation of a transcriptionally repressive state during ZGA and that the repressive state actually dictates the appropriate pattern of gene expression required for further development.

[Chromosome changes of aged oocytes after ovulation].

In mouse, matured oocytes are arrested at metaphase- (M) after ovulation. If the M oocytes are not fertilized on time, they will increasingly become aged in the oviduct. The aging of oocytes can lead to abnormal development of embryos. So it is important to study the mechanism of oocyte aging. Herein, we studied the change of oocyte chromosome after ovulation into the oviduct in vivo. Results indicated that 65% of old oocytes (34 h post hCG) showed aberrant MII, with chromosome misalignment and dispersal, in contrast to the young oocytes (14 h post hCG). In addition, chromosome changes may be associated with the increase of acetylation of histone 3 and histone 4, at lysine 14 and lysine 16 (H3K14 and H4K16), respectively. On the other hand, the decrease of methylation of histone 3 at lysine 9 (H3K9) presumably facilitated aberrant chromosome formation.

[Effects of microsatellite DNA markers on meat quality traits in pig chromosome 13].

In this reseanch, 7 microsatellite DNA loci linked with PPAR gene were selected from the published genetic map of chromosome 13 in pig,and polymorphisms of these microsatellites in 100 samples from Sutai pigs (Duroc x Erhualian) populations were detected. Results revealed that the number of alleles were 6-9, heterozygosity 0.59 - 0.81, polymorphism information content 0.51 - 0.76. Effects of S0021, SW1937, SW482, S0222, S0293, S0281 and SWR2054 on meat quality traits were analyzed with PROC GLM of SAS. Results showed that the effects of S0021 on pH value and SW937 on water-holding capacity reached a significant level at P < 0.01 respectively. The effect of S0293 on tenderness and SW482 on BFT were also significant (P < 0.05). S0222, S0281 and SWR2054 had no significant effect on the 7 selected meat qualitytraits (P>0.05).