• Korean Journal of Animal Reproduction
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• 제21권2호
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• pp.123-130
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• 1997

Normal maturation of the mammalian oocytes is prerequisite for the fertilization and the early embryonic development. We have been tested the effects of purine and its de novo synthetic inhibitor, azaserine(Aza) on the maturation of germinal vesicle(GV) and germinal vesicle breakdown(GVBD) mouse oocytes. Denude-immature oocytes were cultivated in the media containing adenosine, guanosine, and/or azaserine, and checked the matruation stage by monitoring the prominent morphological changes. In GV stage oocytes, GV was arrested temporarily by the adenosine(1.0%) and protractedly by the guanosine(65.9%, P<0.001). The regression was increased significantly at the adenosine(90%, P<0.001) but decreased at the guanosine(1.6%, P<0.05). Inhibiting the de novo synthesis of purine, nuclear maturation rate was increase(90.4% : 96.7%), but GV arrest was significantly increased by cotreatment with guanosine(P<0.001). Polar body extraction significantly was increased at the Aza(P<0.05), but not in others. In GVBD oocytes, adenosine itself did not affect GVBD arrest. Guanosine, on the other hand, elevated GVBD arrest rate(P<0.001), but co-treated with Aza, decreased GVBD arrest(P<0.001). Aza increased GVBD arrest rate(20.2%, P<0.05) compared with control. From those results, we know that guanosine shows more prominent effect on the inhibition of nuclear maturation at the GV stage, and of the 1st polar body extrusion at the GVBD stage. Adenosine showed the cytoplasmic toxicity at GV stage oocyte. Our data speculate that cytoplasmic cAMP level is auto-regulated by endogenous adenylate cyclase while GVBD is inhibited by guanosine, since purine toxicity is not observed in the GVBD stage. And it is showed that purine metabolism is concerned with nuclear maturation, that the amounts of purine metabolism is not even during the oocyte maturation.

• Korean Journal of Animal Reproduction
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• 제27권1호
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• pp.35-44
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• 2003

The present study was performed to investigate the first polar body(PB) extrusion during in-vitro maturation(IVM) and to examine the effect of different maturation time on the embryo development of Korean Native Cows(KNC) with regard to blastocyst(BL) cell numbers and pregnancy rates. PB extrusion did not take place for the first 12 hours(hr) of IVM, and most of KNC oocytes extruded PB from 14 to 20 hr after the onset of maturation. There was no significant difference in cleavage and 8-cell stage rates among the treatment groups, but BL and BL/8-cell rates were significantly higher(P<0.05) in 18 hr maturation group(31.0$\pm$5.7 and 82.0$\pm$5.1%) than 22 and 24 hr maturation group. The proportion of BL formed on day 7 and 8 was significantly higher(P<0.05) in 18 hr maturation group(85%) than in 24 hr maturation group(55%). There was a significant difference(P<0.05) in inner cell mass, trophectoderm and total cell number between day 7 BL produced by in-vivo and IVM 18 hr and day 8 BL produced by IVM 18 hr and 24 hr. Pregnancy rates are also significantly higher(P<0.05) in in-vivo(56.3%) and IVM 18 hr day 7(50.0%) group than day 8 treatment groups(18 hr: 16.7%, 24 hr: 10.5%). These results suggest that KNC oocytes achieve developmental competency within 20 hr of IVM, and "short" IVM (18 hr) is more effective than "long" IVM(24 hr) in embryo development rates, BL cell numbers and pregnancy rates.

• Proceedings of the KSAR Conference
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.66-66
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• 2001

Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.

• Clinical and Experimental Reproductive Medicine
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• 제28권2호
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• pp.111-120
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• 2001

Objective: To verify the expression of leptin receptor (OB-R) in oocytes and preimplantation embryos, the involvement of mitogen activated protein kinase (MAPK or Erk1/2) in the leptin signaling, and effect of leptin on the oocyte maturation in mice. Method: RT-PCR analysis of OB-R was conducted in germinal vesicle (GV)-intact and MII stage oocytes, and 1, 2, 8-cell embryos and blastocysts. Germinal vesicle breakdown (GVB), polar body extrusion, monitored in the presence or absence of leptin ($1{\mu}M$). Following the leptin treatment, temporal changes in MAPK activity were verified by immunoprecipitation and in vitro kinase assay in MII oocytes. Results: The expression of OB-R mRNA was found in GV and MII oocyte but not in the embryos. MAPK activity of the MII oocytes was significantly increased by brief incubation in the HTF supplemented with leptin ($1{\mu}M$). Priming of PD098059, a MEK inhibitor to leptin treatment attenuated the activation of MAPK by leptin in MII oocytes. Following 24 hrs of culture of the GV oocytes, leptin significant increased the GVB and 1 st polar body extrusion. Conclusion: This result suggested that functional interaction between leptin and OB-R resulted in potentiation of MAPK (Erk1/2) activity in MII oocytes through MEK activation and that leptin might be a local regulator of meiotic maturation of the mouse oocytes.

• Clinical and Experimental Reproductive Medicine
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• 제38권4호
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• pp.234-237
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• 2011

Objective: During stimulated IVF cycles, up to 15% of oocytes are recovered as immature. The purpose of this study was to investigate the trend of oocyte maturity in repeated ovarian stimulation for IVF. Methods: One hundred forty-eight patients were selected who underwent two consecutive IVF cycles using same stimulation protocol during 2008 to 2010. Ovarian stimulation was performed with FSH and human menopausal gonadotropin and flexible GnRH antagonist protocol in both cycles. Oocyte maturity was assessed according to presence of germinal vesicle (GV) and the first polar body. Immature oocyte was defined as GV stage or metaphase I oocyte (GV breakdown with no visible polar body) and cultured up to 48 hours. If matured, they were fertilized with ICSI. Results: Percentages of immature oocytes were 30.8% and 32.9% ($p$=0.466) and IVM rates of immature oocytes were 36.2% and 25.7% ($p$=0.077), respectively. A significant correlation was noted between percentage of immature oocytes in the two cycles (R=0.178, $p$=0.03). Women with >40% immaturity in both cycles (n=21) showed lower fertilization rate of $in$ $vivo$ matured oocytes (56.4% vs. 72.0%, $p$=0.005) and lower pregnancy rate (19.0% vs. 27.1%, $p$=0.454) after the second cycle when compared with women with <40% immaturity (n=70). In both groups, female age, number of total retrieved oocyte and embryos transferred were similar. Conclusion: In repeated ovarian stimulation cycles for IVF, the immature oocyte tended to be retrieved repetitively in consecutive IVF cycles.

• Journal of Embryo Transfer
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• 제25권2호
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• pp.97-101
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• 2010

Intracytoplasmic sperm injection (ICSI) is one of the artificial fertilization methods when only a few sperm are available for insemination, and an important tool for the preservation of genetic materials of endangered animal species, especially the male is infertile. Different from other species such as mice and pigs, the conventional ICSI method which uses spiked pipette for injection (Spike-ICSI) is exhibited low success rates in cattle because the bovinesperm head membrane is hard to break during injection procedure. We chose piezo-assisted ICSI (Piezo-ICSI) for the improvement of the injection procedure including sperm head membrane rupture and efficient puncture of the plasma membrane of the oocytes. In this experiment, we compared the efficacy of the bovine ICSI embryo production between the Piezo-ICSI and Spike-ICSI. The second polar body extrusion, pronuclear formation, cleavage and blastocyst formation were evaluated after implementation of two different ICSI techniques. The Piezo-ICSI tended to show comparably higher rates of the second polar body extrusion (41.7%), the pronuclei formation (42.9%) and the two-cell cleavage (41.4%) than Spike-ICSI does (33.3%, 28.6% and 23.5%, respectively) although there is no statistic significance between two groups. In addition, the blastocysts were only obtained from the Piezo-ICSI group (10.3%). Our finding shows that the Piezo-ICSI may be used as an artificial fertilization method in cattle when in vitro fertilization is not applicable.

• Korean Journal of Animal Reproduction
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• 제26권2호
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• pp.165-171
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• 2002

Follicular fluid (FF) contains an oocyte maturation inhibitor with unknown chemical properties. This study was carried out to chemically define the factor(s) inhibiting cumulus cell denudation (CD) and first polar body formation (PBF). Porcine FF (PFF) was extracted with methanol and the extract was serially separated using gel filtration on Superose 12 and Superdex columns. A Superdex fraction was derived with PITC and analyzed with an amino acid analysis column. The results obtained are as follows; PFF had an activity inhibiting both CD and PBF of porcine primary oocytes. Superdex fractions RV2.11 prepared from PFF exhibited an activity inhibiting CD and PBF. By amino acid analysis, the fraction RV2.11 appeared to be proline having the same activity inhibiting CD and PBF. In conclusion, PFF had oocyte maturation inhibitors, of which proline should inhibit CD and PBF.

• Bulletin of the Korean Chemical Society
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• 제35권8호
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• pp.2335-2341
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• 2014

Chemical investigation on the methanol extract of the starfish Ctenodiscus crispatus resulted in the isolation of five steroids, (22E,$24{\zeta}$)-26,27-bisnor-24-methyl-$5{\alpha}$-cholest-22-en-$3{\beta}$,5,$6{\beta}$,$15{\alpha}$,25-pentol 25-O-sulfate (1), (22E,24R,25R)-24-methyl-$5{\alpha}$-cholest-22-en-$3{\beta}$,5,$6{\beta}$,$15{\alpha}$,25,26-hexol 26-O-sulfate (2), (28R)-24-ethyl-$5{\alpha}$-cholesta-$3{\beta}$,5,$6{\beta}$,8,$15{\alpha}$,28,29-heptaol-24-sulfate (3), (25S)-$5{\alpha}$-cholestane-$3{\beta}$,5,$6{\beta}$,$15{\alpha}$,$16{\beta}$,26-hexaol (4), and ${\Delta}7$-sitosterol (5). Their structures were identified by extensive spectroscopic analyses, including 1D, 2D NMR and MS and chemical methods. Compound 4 showed cytotoxicity against human hepatoma HepG2 and glioblastoma U87MG cells via inhibition of cell growth and induction of apoptosis. Induction of apoptosis by 4 was demonstrated by cell death, DNA fragmentation, increased Bax/Bcl-2 protein ratio and the activation of caspase-3, caspase-9 and poly (ADP-ribose) polymerase (PARP).

• Journal of computational fluids engineering
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• 제20권1호
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• pp.47-56
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• 2015

In this paper, we study the effect of various turbulence models by comparing the aerodynamic characteristics and the flow patterns computed for aircraft models. An in-house CFD solver, MSAPv, that solves the three dimensional RANS equations with the turbulence model equations is used. The turbulence models used in this study are the Spalart-Allmaras model, Menter's $k-{\omega}$ SST model, Coakley's $q-{\omega}$ model, and Huang and Coakley's $k-{\varepsilon}$ model. DLR-F6 WB and WBNP configurations are selected for the study. We concentrate on the separated flow pattern variations with the turbulence models at the wing-body junction and the wing-pylon junction as well as drag polar curves.

• Asian-Australasian Journal of Animal Sciences
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• 제13권7호
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• pp.995-1002
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• 2000

To evaluate energy efficiency and partition of nutrients, 32 piglets were weaned at 14 d of age and individually fed diets containing 15% fat from coconut oil (CO, medium chain saturated), high oleate sunflower oil (HOSO, n-9 series), soybean oil (SO, n-6 series), or linseed oil plus fish oil, (LF, n-3 series). After 4 weeks, the subjects were sacrificed to evaluate empty body composition and apparent ileal digestibility with the slaughter method. No statistical effect of dietary fat sources on growth was observed. The digestibility of fat from the coconut oil diet was higher than fats from the diets containing high levels of unsaturated fatty acids. The efficiency of use of metabolizable energy for growth averaged 63% and was not affected by the diet. Dietary fat composition was reflected strongly in backfat. Total body neutral and polar fatty acids were influenced too. For the whole body phospholipid fraction the ratio of n-6 to n-3 and the double bond index were 4.3, 5.8, 7.2, 0.78 and 69, 87, 89, 87 for CO, HOSO, SO, and LF respectively. These results show that for the coconut oil diet the degree of unsaturation of phospholipids in the body was lower and that, in the other diets, it did not differ, but double bond index was maintained with different n-6 to n-3 ratios in carcass fat. On the whole the data on body fat composition indicate that the dietary fat tended to be deposited in similar quantity in the body, whatever was the dietary fatty acid profile.