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Abstract.Objective and Design:??The role of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, was tested using a mouse asthma model.Materials:??One hundred and four male BALB/c mice were used in this study.Treatment:??Mice were actively sensitized with an intraperitoneal injection of ovalbumin (OVA) and challenged with repeated nebulization of 1 w/v% OVA. Polyclonal anti-MIF antibody was intraperitoneally injected at 10 mg/kg during the antigen challenge period.Methods:??Bronchoalveolar lavage (BAL) was performed 8 h after the last challenge. Airway hyperresponsiveness to inhaled methacholine was measured 24 h after the last challenge.Results:??Antigen challenge to immunized mice induced increase in inflammatory cells and concentration of Th2 cytokines in BAL fluid (BALF), and caused the development of airway hyperresponsiveness. Anti-MIF antibody significantly decreased the numbers of inflammatory cells including macrophages, eosinophils, lymphocytes and neutrophils in BALF from OVA-challenged mice. Prednisolone decreased the numbers of eosinophils, lymphocytes and neutrophils but not macrophages. Anti-MIF antibody reduced airway hyperresponsiveness. Anti-MIF antibody affected neither the cytokine levels in BALF nor the IgE levels in serum.Conclusion:??MIF was involved in the antigen-induced inflammatory cell accumulation in the lung and airway hyperresponsiveness without affecting immune responses. [ABSTRACT FROM AUTHOR]

Perturbation of circadian rhythm is believed to be detrimental to the physiology of organs, including the mammalian ovary. However, the molecular mechanisms that are regulated by circadian rhythm in the ovary have not been identified. To identify the molecular mechanisms that are regulated by circadian rhythm and to speculate on the physiologies that are likely to be damaged by perturbation of circadian rhythm in the ovary, we searched for genes in which the amount of transcripts oscillates every 24 h in the mouse ovary. To achieve this, expression profiles of circadian genes (per1, per2, and bmal1) that code transcription-regulation factors for which transcription activities are known to oscillate every 24 h in almost all organs, and wee1, the transcription activity of which circadian genes regulate and which is known to elongate the G2 phase in the cell cycle, were analyzed in this study. Six-week-old female ICR mice were kept individually under a lighting schedule with lights on for 14 h followed by lights off for 10 h. A vaginal smear of each mouse was collected every day to determine its estrous cycle. Ovaries of 3 mice were collected continuously every 4 h over a 4-day period from the start of the light period on the day of proestrus. Total RNA was extracted from each ovary, and 500 ng each was used for cDNA synthesis. Transcripts of each gene and of tbp were quantified by real-time PCR, and the amount of the transcripts of each gene in each sample was divided by the amount of tbp transcripts. The obtained relative values in each sample were used as the representative data of the amount of transcripts of each gene. The amounts of per1, per2, and bmal1 clearly oscillated every 24 h. The maximum and minimum values of per1 and per2 were observed at 16 and 4 h, respectively, after onset of the light period each day. The maximum and minimum values of bmal1 were observed at the time of onset of the light period and at 12 h after onset of the light period each day. Averages of the maximum values of per1, per2, and bmal1 each day were significantly greater than averages of the minimum values (per1, 3.60 0.10 and 1.38 0.09; per2, 0.82 0.08 and 0.27 0.06; bmal1, 0.61 0.05 and 0.17 0.01; P < 0.05). The cyclicity in the oscillation of the amount of wee1 transcripts was weaker than that observed in circadian genes, but the average of values that were obtained from 12 to 20 h after onset of the light period each day was significantly greater than that obtained from 0 to 8 h (0.29 0.02 and 0.22 0.01; P < 0.05). Our results suggested that the cell cycle of ovarian cells is regulated in a circadian manner through wee1 transcription, which is regulated by circadian genes of which the amounts of transcripts oscillate every 24 h. Because an abnormal cell cycle seems to trigger the development of tumors or follicular cysts, perturbation of circadian rhythm may cause those ovarian diseases. [ABSTRACT FROM AUTHOR]

Zygotic gene activation (ZGA) starts at the G2 phase at the 1-cell stage in the mouse. However, the molecular mechanism of ZGA has not been completely elucidated. We have investigated the molecular functions of many gene clusters, DD clones obtained by differential display assays for ovulated eggs at the M II stage and 1-cell stage embryos at the G2 phase. As a result, we have identified a functionally unknown gene, whose sequence did not match a known transcript in the gene bank DD2-2 gene. Here, we report the expression profile and knockdown analysis of the DD2-2 gene in mouse pre-implantation embryos. Nucleotide sequence analysis of the DD2-2 cDNA revealed that the open reading frame of 1056 bp encodes a protein of 351 amino acids with a predicted molecular mass of 41.5 kDa. The deduced amino acid sequence indicated that DD2-2 protein might be a soluble protein without a signal peptide. We first investigated the expression profiles of DD2-2 in pre-implantation embryos by quantitative real-time PCR using an ABI PRISM 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). To investigate the effect of knockdown of the DD2-2 gene on the development of pre-implantation embryos, we injected p?-act/antisenseDD2-2/IRES/EGFP into male pronuclei of embryos at 7 to 9 h after insemination (hpi) and observed the development of embryos that showed EGFP expression at 24 hpi. Real-time PCR analysis of pre-implantation embryos showed that maternal DD2-2 mRNA at a low level significantly increased up to the early 2-cell stage, and significantly decreased by the 4-cell stage and later, suggesting that DD2-2 gene specifically expresses at major ZGA. In the knockdown analysis, EGFP-positive embryos with p?-act/antisenseDD2-2/IRES/EGFP showed a lower rate of development to the 4-cell stage and later, compared with that of EGFP-positive embryos with p?-act/luc+/IRES/EGFP [72% (94/130) vs. 54% (71/131); P < 0.05], indicating that the knockdown of DD2-2 by antisense RNA resulted in a inhibition of pre-implantation development. In conclusion, the DD2-2 gene, a functionally unknown gene, may play an important role in pre-implantation development.This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST. [ABSTRACT FROM AUTHOR]

We report extragalactic observations of two fluorine-bearing species, hydrogen fluoride (HF) and fluoronium (H2F+), in the z = 0.89 absorber in front of the lensed blazar PKS 1830−211 with the Atacama Large Millimeter/submillimeter Array. HF was detected toward both southwest and northeast images of the blazar, with column densities >3.4 × 1014 cm−2 and 0.18 × 1014 cm−2, respectively. H2F+ was not detected, down to an upper limit (3σ) of 8.8 × 1011 cm−2 and an abundance ratio of [H2F+]/[HF] 1/386. We also searched for H2F+ toward the Galactic sources NGC 6334 I and W51C, and toward Galactic center clouds with the Herschel HIFI spectrometer.6 The upper limit on the column density was derived to be 2.5 × 1011 cm−2 in NGC 6334 I, which is 1/68 of that for H2Cl+. In constrast, the ortho transition of H2Cl+ is detected toward PKS 1830–211. To understand the small abundance of interstellar H2F+, we carried out laboratory experiments to determine the rate constants for the ion–electron recombination reaction by infrared time-resolved spectroscopy. The constants determined are ke(209 K) = cm3 s−1 and cm3 s−1 for H2F+ and H2Cl+, respectively. The difference in the dissociative recombination rates between H2F+ and H2Cl+ by a factor ∼2 and the cosmic abundance ratio [F]/[Cl] ≈ 1/6 are not enough to explain the much smaller abundance of H2F+. The difference in the formation mechanism of H2F+ and H2Cl+ in interstellar space would be a major factor in the small abundance of H2F+. [ABSTRACT FROM AUTHOR]

Histone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genomes after fertilization or of transferred cell genomes after nuclear transfer to establish a totipotent state for normal development. In the fertilization of bovine oocytes, asynchronous histone acetylation occurs during pronuclear formation in the manner that modification of the paternal genome precedes that of the maternal genome (Wee et al. 2006 J. Biol. Chem. 281, 6048–6057). In the present study, the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, during in vitro fertilization (IVF) of bovine oocytes on subsequent embryonic development were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse ovaries were in vitro-matured (IVM) for 21 h in TCM-199 supplemented with 5% v/v FCS, 0.5 mM sodium pyruvate, 0.02 AU mL-1 FSH, and 1 g mL-1 estradiol-17? at 39C under 5% CO2 in air. After IVM, the oocytes were subjected to IVF with 3 106 mL-1 of Percoll gradient-selected sperm in a defined medium (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) supplemented with 0 (control), 5, 50, and 500 nM TSA for 18 h. After IVF, presumptive zygotes were freed from cumulus cells and cultured in mSOF medium until 168 h post-insemination (hpi) at 39C under 5% CO2, 5% O2, and 90% N2 with high humidity. Cleavage and blastocyst development were assessed at 48 and 168 hpi, respectively. Inner cell mass (ICM) and trophectoderm (TE) of blastocysts were differentially stained by the method of Thouas et al. (2001 Reprod. Biomed. Online 3, 25–29) to assess cell number and ICM/TE ratio. Experiments were replicated 3 times. Data are presented as means SEM and statistically analyzed by multiple comparison with the Holm method. Rates of cleavage (0 nM: 71.0 7%, n = 102; 5 nM: 75.5 5%, n = 106; 50 nM: 68.8 6%, n = 105; and 500 nM: 71.7 4%, n = 98) and blastocyst formation (21.4 5%, 22.3 6%, 17.8 2%, and 18.2 2%, respectively) were similar among the groups. However, 500 nM TSA significantly (P < 0.05) increased ICM and total cell numbers (59.8 4 and 143.5 7, respectively, n = 31) compared with the control (43.1 3 and 120.9 7, n = 31). In addition, ICM/TE ratios were higher in the 50 nM (0.81 0.08, n = 29) and 500 nM (0.92 0.2, n = 31) groups than in the control (0.59 0.04, P < 0.05). These results suggest that TSA treatment during IVF of bovine oocytes does not affect the blastocyst rate but alters the cell numbers and their allocation to ICM and TE. Overriding epigenetic modification of the genome during fertilization may have a carryover effect on cell proliferation and differentiation in pre-implantation embryos.This study was supported by a grant from Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST. [ABSTRACT FROM AUTHOR]

Maternal transcripts that accumulate during oocyte growth are involved in the meiotic maturation, the initiation of the first mitosis, and the later pre-implantation development. Although the conserved E-box sequences in promoter region of some maternal genes (for example, Zp3 and Gdf-9), which are important in regulating gene transcripts as binding sites of transcriptional factors, may play a role in the oocyte-specific expression in ovary, the molecular mechanism that regulates the expression of the maternal genes is still not known. In this study, we have focused on the transcriptional activity of promoter regions to clarify the molecular mechanism of transcriptional regulation of these maternal genes [Histone H1oo (H1oo), Nucleoplasmin 2 (Npm2), and Zygote arrest 1 (Zar1)]. First, we observed the expression of firefly luciferase expression vectors under promoter regions of 3 maternal genes in oocytes isolated from 10- to 12-day-old mice, which is mainly NSN-type, transcriptionally active form (Bouniol-Baly et al. 1999 Biol. Reprod. 60, 580–587). Transcriptional activities of H1oo (-3975), Npm2 (-2610), and Zar1 (-5187) promoters were detected in oocytes, the relative luciferase activities being an average of 70, 130, and 12, respectively. On the other hand, these promoter activities were not detected in embryos at the 2-cell stage. Furthermore, deletion analysis of promoter elements (E-boxes) of H1oo and Npm2 was done by microinjecting deletion constructs into oocytes. In the H1oo promoter, deletion of sequences between -3975 and -72 bp from the transcription start site resulted in one-third of the level obtained in the full H1oo (-3975) promoter. In addition, deletion of sequence -68 bp resulted in no detection of luciferase activity. These findings indicate that the putative distal promoter sequences exist at the 52-flanking region (-3975 to -759) of the H1oo gene and that the region (-314 to -68) including the E-box region (-72) may be required for high-level transcriptional activity of the H1oo promoter. In the Npm2 promoter, deletion of sequences between -2610 and -180 bp from the transcription start site resulted in one-third of the level of wild-type activity of the Npm2 (-2610) promoter. In addition, deletion of sequence -101 bp resulted in no detection of luciferase activity. These findings also indicate that 3 putative distal promoter sequences exist at the 52-flanking region (-2610 to -210) of the Npm2 gene and that the region (-210 to -101) that includes the E-box region (-180) is crucial for high-level transcriptional activity of the Npm2 promoter. In conclusion, the E-box may be a key regulatory region for the expression of two of the maternal genes (H1oo and Npm2) examined. Currently, we are attempting to identify the transcriptional factor binding sites by DNase I footprint analysis and gel mobility shift assay. [ABSTRACT FROM AUTHOR]

It has been reported that activity of Rho, one of the GTPases, is essential for division of nuclei and cytoplasm of fertilized mouse eggs. Since it has been reported that alteration of activities of GTPases modifies their ability to attach to each of their effector proteins in somatic cells, effector proteins seem to be able to control not only progression but also repression of cell division by changing their cellular localizations through activities of GTPases. For this reason, Rhophilin-2, one of the effector proteins of Rho, seems to be involved in the decision of progression of division of fertilized mouse eggs. To examine whether this involvement works in fertilized mouse eggs, cellular localization of Rho and Rhophilin-2 in fertilized mouse eggs that were treated with Rho inhibitor were analyzed. Moreover, cellular localization of GABA A receptor association protein (GABARAP), which was identified in our previous study (Matsuoka et al. 2006 Reprod. Fertil. Dev. 18, 176–177) as a protein that interacts with Rhophilin-2, was also analyzed. Fertilized mouse eggs were obtained from in vitro fertilization technique. One group of fertilized eggs was obtained at 24 h after insemination as experimental control. To obtain the mouse eggs in which Rho activities were inhibited, Clostridium botulinum C3 exoenzyme (C3-CB), an inhibitor of Rho activity, was injected into the other group of fertilized mouse eggs at 12 h after insemination, and were collected after 12 h of subsequent culture. Cellular localization of Rho (n = 100), Rhophilin-2 (n = 10). and GABARAP (n = 10) in the collected oocytes was analyzed by using immunofluorescence. Our results showed that Rho and Rhophilin-2 were co-localized at the midbody microtubule, which is an important device for cytoplasmic division in control eggs. However, the inhibition of Rho activity did not modify the co-localization of Rho and Rhophilin-2. On the other hand, localization of GABARAP was modified by the inhibition of Rho activity, and GABARAP was detected around the nuclei of fertilized eggs in which Rho activity was inhibited. In the next experiment, we examined whether interaction of Rhophilin-2 and GABARAP was modified by the inhibition of Rho activity by using a co-immunoprecipitation assay (co-IP) (n = 100). The interaction of Rhophilin-2 and GABARAP was found to disappear after inhibition of Rho activity. These results suggest that activity of Rho seems to regulate cytoplasmic division through Rhophilin-2 modification. Moreover, Rho seem to modulate the nuclear division of fertilized mouse eggs by regulating the interaction between Rhophilin-2 and GABARAP.This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST. [ABSTRACT FROM AUTHOR]

Cloning by adult somatic cell nuclear transfer (SCNT) has proven to be successful for the production of clones from many species (Keith 2004 Cytogenet. Genome Res. 105, 285). However, somatic cloning is currently highly inefficient. One of the reasons for this is that SCNT is believed to be associated with epigenetic errors including abnormal DNA methylation of the reconstructed embryo. The Oct-3/4 gene, a member of the POU transcription factor family, is expressed throughout the pre-implantation embryo. Abnormal expression of the Oct-3/4 gene in the nuclear-transferred embryo is either directly or indirectly caused by nuclear transfer and is suggested to be indicative of a general failure to reset the genetic program (Boiani et al. 2002 Genes Dev. 16, 1209). In this study, we investigated the DNA methylation profiles of the Oct-3/4 gene in the genome of SCNT embryos, using bisulfite sequencing analysis. Then, we observed the detailed subcellular localization of Oct-3/4 proteins in SCNT embryos using immunocytochemical (ICC) analysis. Nuclear transfer of cumulus cell nuclei was carried out as previously described (Wakayama et al. 1998 Nature 394, 369). After nuclear transfer, embryos were subsequently cultured in KSOM media to the morula and blastocyst stages. We compared the methylation profiles of 3 transcriptional control elements (distal enhancer, DE; proximal enhancer, PE; and promoter) of the upstream region of the Oct-3/4 gene with the genome of in vitro fertilization (IVF) and SCNT embryos. The methylation rate of CpG sites in the DE and promoter regions of both IVF and SCNT embryos was low at both the morula and the blastocyst stages. What''s interesting is that there was a significant difference in the methylation level on CpG sites in the PE element between IVF and SCNT embryos. At the morula stage, the methylation level on CpG sites in the PE element was very low in the IVF embryo and moderately high in the SCNT embryo (0.9% and 26.3%). Conversely, at the blastocyst stage, CpG sites in the PE element showed high methylation in the IVF embryo and low methylation in the SCNT embryo (55.2% and 10.5%). CpG sites in the PE element were lightly methylated (3.0%) in the inner cell mass (ICM) of the IVF embryo. This means that the main portion of methylation in the IVF blastocyst embryo occurred at the trophectoderm (TE). On the other hand, in ICM of the SCNT embryo, the methylation level of each embryonic cell was almost the same in the whole blastocyst embryo (9.8% and 10.5%). As a result, it is highly possible that the CpG sites in the PE element of ICM were methylated as in the TE. ICC analysis revealed that some SCNT embryos showed aberrant Oct-3/4 expression in the TE. These results indicate that the methylation of CpG sites in the Oct-3/4 PE element may be related to expression of Oct-3/4 in the mouse IVF and SCNT embryos. These differences in methylation level between IVF and SCNT embryos were reflected as abnormal expressions of Oct-3/4 on SCNT embryos.This study was supported by the 21st COE Program of MEST. M.K. is a JSPS Research Fellow and supported by Grant-in Aid for Scientific Research (No. 1751132) of JSPS. [ABSTRACT FROM AUTHOR]