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This study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P < 0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P < 0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P < 0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P < 0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P < 0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.
We present high resolution molecular line observations of dusty AGN and starburst in nearby luminous infrared galaxies (LIRGs), VV 114 (band 3/4/7) and NGC 1614 (band 3/6/7/9), with ALMA. Multi-frequency imaging from 4.8 GHz to 691 GHz of NGC 1614 allows us to study spatial properties of the radio-to-FIR continuum and multiple CO transitions, and we find the CO excitation up to Jupp = 6 can be explained by a single ISM model powered by nuclear starbursts. Our processing line imaging survey for VV 114 detected at least 30 molecular lines which show different chemical composition from region to region. Multi-molecule imaging helps us to diagnose the chemical differences of dusty ISM, while multi-transition imaging allows us to investigate gas physical conditions affected by nuclear activities directly.
Our new compilation of interferometric CO data suggests that nuclear and extended molecular gas disks are common in the final stages of mergers. Comparing the sizes of the molecular gas disk and gas mass fractions to early-type and late-type galaxies, about half of the sample show similar properties to early-type galaxies, which have compact gas disks and low gas mass fractions. We also find that sources with extended gas disks and large gas mass fractions may become disk-dominated galaxies.
Ultrasonic spray-assisted mist deposition techniques have been developed as a cost-effective and environmental friendly deposition method for oxide and organic thin films. The chemical vapor deposition (CVD) of a variety of oxide thin films having unique functions, such as Cr2O3, Cu2O, Fe3O4, and Al2O3 thin films, has been demonstrated as well as high-quality ZnO and Ga2O3 films ever reported. In addition to the films deposition by the CVD process, the deposition of organic material thin films from the source solution has also been achieved; as examples we have shown the patterned deposition of water-soluble fluorescent polymers with a metal mask. This may substitute the spin-coating technique and contribute to increase the source consumption efficiency in the thin film deposition. We appeal that the mist deposition is a unique and promising technique as a green chemical route for film deposition.
Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been implicated in the development of normal- and high-tension glaucoma. We investigated the effects of unoprostone on extracellular signal-regulated kinase (ERK) in ET-1-induced retinal ganglion cell (RGC) death and optic nerve injury. Our morphometric study showed that intravitreal injection of ET-1 led to cell loss in the RGC layer (RGCL) in 28 days. Western blot analysis showed decreased neurofilament (NF) protein in the optic nerve 28 days after ET-1 injection. In this in vivo model, increased phosphorylated ERK (p-ERK) was observed in the retina on 1 day and subsequently in the optic nerve from 7 days after ET-1 injection. Simultaneous injection of M1, as a metabolite of unoprostone, showed further increased p-ERK levels compared with ET-1 injection alone. Our morphometric study of flat-mount preparations stained with cresyl violet or retrograde labeling with a neuro-tracer and Western blot analysis of NF showed that inhibition of ERK phosphorylation led to acceleration of ET-1-induced RGC death and optic nerve damage. In addition, M1 significantly attenuated both RGC loss and the decrease in NF protein induced by ET-1. The protective effects of M1 were significantly inhibited by U0126, an ERK inhibitor. These results suggest that unoprostone has neuroprotective effects against ET-1-induced neuronal injury through ERK phosphorylation.
The possibility of using aged porcine oocytes treated with caffeine, which inhibits the decrease in M-phase promoting factor activity, for pig cloning was evaluated. Cumulus–oocyte complexes (COCs) were cultured initially for 36h and subsequently with or without 5mM caffeine for 24h (in total for 60h: 60CA+ or 60CA– group, respectively). As a control group, COCs were cultured for 48h without caffeine (48CA–). The pronuclear formation rates at 10h after electrical stimulation in the 60CA+ and 60CA– groups decreased significantly (p<0.05) compared with the 48CA– group. However, the fragmentation rate was significantly higher (p<0.05) in the 60CA– group than in the 60CA+ and 48CA– groups. When the stimulated oocytes were cultured for 6 days, the 60CA+ group showed significantly lower blastocyst formation and higher fragmentation or degeneration rates (p<0.05) than the 48CA– group. However, the number of total cells in blastocysts was not affected by maturation period or caffeine treatment. When somatic cell nuclei were injected into the non-enucleated oocytes and exposed to cytoplasm for a certain duration (1–11h) before the completion of maturation (48 or 60h), the rate of nuclear membrane breakdown after exposure to cytoplasm for 1–2h in the 60CA– oocytes was significantly lower (p;<0.05) than in the other experimental groups. The rate of scattered chromosome formation in the same 60CA– group tended to be lower (p=0.08) than in the other groups. After the enucleation and transfer of nuclei, blastocyst formation rates in the 60CA+ and 60CA– groups were significantly lower (p<0.05) than in the 48CA– group. Blastocyst quality did not differ among all the groups. These results suggest that chromosome decondensation of the transplanted somatic nucleus is affected by both the duration of exposure to cytoplasm and the age of the recipient porcine oocytes, and that caffeine treatment promotes nuclear remodelling but does not prevent the decrease in the developmental ability of cloned embryos caused by oocyte aging.
Lipid content in mammalian oocytes or embryos differs among species, with bovine and porcine oocytes and embryos showing large cytoplasmic droplets. These droplets are considered to play important roles in energy metabolism during oocyte maturation, fertilisation and early embryonic development, and also in the freezing ability of oocytes or embryos; however, their detailed distribution or function is not well understood. In the present study, changes in the distribution and morphology of porcine lipid droplets during in vivo and in vitro fertilisation, in contrast to parthenogenetic oocyte activation, as well as during their development to blastocyst stage, were evaluated by transmission electron microscopy (TEM). The analysis of semi-thin and ultra-thin sections by TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated with mitochondrial aggregates in the oocytes, irrespective of whether the oocytes had been matured in vivo or in vitro. Immediately after sperm penetration, the electron density of the lipid droplets was lost in both the in vivo and in vitro oocytes, the reduction being most evident in the oocytes developed in vitro. Density was restored in the pronculear oocytes, fully in the in vivo specimens but only partially in the in vitro ones. The number and size of the droplets seemed, however, to have decreased. At 2- to 4-cell and blastocyst stages, the features of the lipid droplets were almost the same as those of pronuclear oocytes, showing a homogeneous or saturated density in the in vivo embryos but a marbled or partially saturated appearance in the in vitro embryos. In vitro matured oocytes undergoing parthenogenesis had lipid droplets that resembled those of fertilised oocytes until the pronuclear stage. Overall, results indicate variations in both the morphology and amount of cytoplasmic lipid droplets during porcine oocyte maturation, fertilisation and early embryo development as well as differences between in vivo and in vitro development, suggesting both different energy status during preimplantation development in pigs and substantial differences between in vitro and in vivo development.
Culturing of matured porcine oocytes in vitro results in the enhancement of their cytoplasmic ability for oocyte activation (so-called ageing), although they are arrested at metaphase II. The enhanced ability for oocyte activation is related to decreased activity of the maturation promoting factor (MPF). In the present study we clarified the molecular mechanism of MPF inactivation during ageing, especially the changes in the phosphorylation status of p34cdc2, a catalytic subunit of MPF, compared with that in fertilised oocytes. The MPF activity decreased gradually when maturation culture was prolonged from 36 to 72 h, confirming the decreasing MPF activity in aged oocytes. The activity of 48 h matured oocytes also decreased after in vitro fertilisation. Immunoblotting of p34cdc2 with anti-PSTAIRE antibody revealed that the culturing of matured oocytes induces a gradual increase in pre-MPF, which is a p34cdc2 and cyclin B complex inactivated by phosphorylation at the inhibitory phosphorylation site of p34cdc2. In contrast, pre-MPF decreased after fertilisation, indicating the degradation of cyclin B. These results suggest that the molecular mechanisms of inactivation of MPF are different between oocyte activation and ageing, and that the mechanism during ageing might be based on the inhibitory phosphorylation of p34cdc2, whereas that of oocyte activation is based on the degradation of cyclin B.
Hydroxyapatite (HAP : Ca10(PO4 ) 6 (OH) 2 ) and aluminosili-cate were easily synthesized using a hydrothermal process with mixing ratios of Ca/P=1.67 (Ca(PO3)2 and Ca(OH)2) and Si/Al=4 (SiO2 and Al(OH)3). The temperature was kept constant at 573 K for 60 minutes. .Cesium (Cs) and Cerium (Ce) contained in Ca(PO3)2 , were found to take on a stable form in the pollucite (CsAlSi2O6) structure and the monazite (CePO4) structure.
The dependences of dopant species and concentrations on the growth of oxide precipitates have been studied using transmission electron microscopy. Doping species are phosphorus, antimony and boron. Samples were annealed at 800°C and 850°C for 24∼384hr in dry nitrogen. In phosphorus-doped silicon, the precipitate density is independent of doping concentration and the growth of precipitate obeys the three-quarter power law. The enhancement of the precipitate growth is observed in antimony-doped silicon. On the other hand, the precipitate growth is suppressed in heavily boron-doped silicon as compared with that of lightly boron-doped silicon. This indicates the generation of excess silicon interstitials in heavily boron-doped silicon.
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