• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.47-57
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• 2000

Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL(표현불가)/CBA(표현불가)). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. Results: The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group ($50.2{\pm}14.0$) than in 6/8 ($26.5{\pm}6.2$), 5/8 ($25.0{\pm}5.5$), and 4/8 ($17.8{\pm}7.8$) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group ($25.9{\pm}10.2$), compared with the control ($50.2{\pm}14.0$), 7/8 ($56.0{\pm}22.2$), and 6/8 ($55.3{\pm}25.5$) groups. Conclusion: After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.

• The Korean Journal of Zoology
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• v.13 no.3
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• pp.68-74
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• 1970

In the previous studies, the present author found that high proportion of the follicular oocytes from mouse and rabbit ovaries are able to resume their maturation division in the anterior chamber of the eye in which they have been incubated by auto- or homoplastic transplantation. Especially in the case of the homoplastic transplantation, it was known that no trouble has been detected in the process of resumption of the oval maturation in particular connection with the antigen-antibody reaction between donor and recipient. These findings provide a possibility that the follicular oocytes from various animals would be matured in the eye even after the xenoplastic transplantation. Under such an assumption, the present studies were performed to examine the behavior of the follicular oocytes in the eye chamber of the animals of different species. For the donor of the follicular oocytes, domestic rabbits, albino rats of Sprague-Dowley strain, and albino mice of A-strain bred in our laboratory were used. The oocytes obtained from the ovarian follicules were introduced to the anterior chamber of the eye of different species of animals, with an exception of rabbit in which only the female animals were used as a recipient. The procedures of collection of ova, introduction to the eye, harvest from the eye ball, fixation, and staining were the same as mentioned in the previous reports (Cho, 1967b; Cho and Kim, 1968). The conclusions obtained are summarized as below. 1. The rabbit follicular oocytes are able to mature in the eye chambers of both male mouse and rat, although the proportion of the maturation is lower than when they are incubated autoplastically in the eye. When the ova were incubated in the male mouse eye for 24 hours, 21 per cent of them showed chromosomes at metaphase I and II, whereas the rate was 32 per cent when they were incubated in the eye of the male rat. These are apparently low comparing to the rate of 52 per cent of autoplastic transplantation. 2. When rat follicular oocytes were transferred into the mouse eye chamber and recovered after 24 hours, 43 per cent of them produced the mataphase I and II chromosomes. This proportion was higher than the result of the homoplastic transplantation which yielded 23 per cent of the ova on maturation. 3. The most striking result was found in the experiment with mouse follicular oocytes. Seventy-six per cent of the oocytes resumed their maturation division within 24 hours after they were transferred into the male rat eye chamber, and this figure was significantly high compared to the result o 55 per cent obtained by the homoplastic transplantation. In the rat eye, the induction of the degenerative ova also was low (19%). On the other hand, the proportion of the oval maturation decreased to 45 per cent, while that of degeneration increased 33 per cent when they were incubated in the eye of the female rabbit. 4. It was apparent from the present experiments that the follicular oocytes can reveal their activation to maturation in the eye chamber which contains aqueous humor which is known to be composed of low protein content and of very little gamma-globulin which acts as an antibody(Oser, 1965), and that it shows higher osmolarity than blood serum(Levene, 1958). Taking these properities into consideration the humor may provide unfavourable environment to the cells and tissues incubated in. However, it could be noteworthy finding that only the follicular oocytes in the eye of the different species can grow in healthy condition although the maturation rates are varied with the animal species. The fact that the rabbit follicular oocytes show the lower proportion in maturation may be due to the greater amount of the yolk granules in the egg cytoplasm than those in the mouse and rat oocytes. That the mouse oocytes incubated in the eye of the rat mouse and rat oocytes. That the mouse oocytes incubated in the eye of the rat resumed their maturation process in greater proportion would e explained by the fact that the rat eye chamber particularly provides the better environment to the mouse oocytes than the eye chamber of mouse does.

• The Korean Journal of Nuclear Medicine
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• v.34 no.4
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• pp.344-352
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• 2000

Purpose: Re-188-Hydroxyethylidene diphosphonate (HEDP) is a new cost-effective agent for systemic radioisotope therapy of metastatic bone pain. We investigated the influence of carrier for labeling and biodistribution of Re-188-HEDP using HEDP kit with or without carrier ($KReO_4$). Materials and Methods: The kits (HEDP 15 mg, gentisic acid 4 mg and $SnCl_2.2H_2O_2$ 4.5 mg) with or without carrier ($KReO_4$ 0.1 mg) were labeled with Re-188 solution, made available from an in-house generator by boiling for 15 min. We compared the labeling efficiency and stability of carrier-added and carrier-free preparations of Re-188-HEDP Biodistribution and imaging studies of each preparation were performed in ICR mice ($1.85{\sim}3.7MBq/0.1ml$) and SD rats ($74.1{\sim}85.2MBq/0.5ml$). Results: The carrier-added preparation showed high labeling efficiency (95% at pH 5) and high stability in serum (88%, 3 hr). However, the carrier-free preparation showed low labeling efficiency (59% at pH 5) and low stability (43%, 3 hr). The carrier-added preparation showed high uptake in bone and low uptake in stomach and kidneys. However, the carrier-free preparation showed lower uptake in bone and higher uptake in both stomach and kidneys, which is supposed to be due to released perrhenate. The carrier-added preparation also showed better images with higher skeletal accumulation, lower uptake in other organs and lower soft tissue uptake than the carrier-free preparation. Conclusion: The results of these studies clearly demonstrate that addition of carrier perrhenate is required for high labeling efficiency, stability, bone uptake and good image quality of Re-188-HEDP.

• Journal of Life Science
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• v.25 no.3
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• pp.307-316
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• 2015

Beetle larvae have been used as a traditional medicine to treat various human liver diseases. To prove the liver protective function of Allomyrina dichotoma larvae (ADL), we induced liver damage by the intraperitoneal injection of a hepatotoxic reagent, diethylnitrosamine (DEN), to C3H/HeN male mice and orally administered freeze-dried ADL powder. ADL powder lessened DEN-induced hepatotoxicity considering the reduced signs of acute and chronic hepatotoxicities, such as the ALP level in the blood serum, TUNEL-positive hepatocytes, ductural reactions, steatotic hepatocytes, and collagen deposition of the Masson’s trichrome staining. In addition to hepatoprotection, the anti-cancer activity of ADL has been examined. The ADL powder was extracted with ethanol and then fractionated with hexane, ethyl acetate, and water by a solvent partition technique. The ethyl acetate fraction showed cytotoxicity to various cancer cells through induction of apoptosis and necrosis, as well as the perturbed metabolism of the cancer cell to trigger autophagy. Collectively, ADL contains bioactive substances that can protect hepatocytes from toxic chemicals and trigger cell death in cancer cells. Thus, further purification and analyses of ADL fractions could lead to the identification of novel bioactive compounds.

• Applied Microscopy
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• v.7 no.1
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• pp.21-43
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• 1977

This experiment was undertaken in order to localize the labeled dbcAMP (dibutyryl cyclic AMP) in oocytes whose development has been suppressed by cold dbcAMP for 6 or 19 hours in vitro. Mouse oocytes were obtained from the ovaries of 3-4 week old A strain female mice, by puncturing the Graafian follicles in the modified Krebs-Ringer bicarbonate salt solution under the dissecting microscope. Those oocytes which have intact germinal vesicle were cultured in the basic culture medium supplemented with 0.4% bovine serum albumin (BSA). Cultivation of the oocytes was carried out in a microtube developed by Cho (1974). The cultures were then incubated in a humidified 5% $CO_2$ incubator maintained at $37^{\circ}C$ for 6 or 19 hours (Donahue, 1968). DbcAMP was added to culture medium for a final concentration of 100ug/ml, and $^3H-dbc$ AMP (specific activity 13 Ci/mM) for a final concentration of $40{\mu}Ci/ml$ was also added to the medium. For electron microscopic autoradiography, those oocytes recovered from the culture were washed with phosphate buffer (pH 7.4), and immediately prefixed in a 2.5% glutaraldehyde overnight and postfixed for 2 hours at $4 ^{\circ}C$ in 1% osmium tetroxide in phosphate buffer with pH 7.4 (Palade, 1952). After fixation, the materials were dehydrated in graded alcohol series and embedded in Epon 812 mixture based on the standard procedures (Luft, 1961). The thin sections $600-700{\AA}$ thick were mounted on the grids of 200 meshes. The grids containing sections were coated with a nuclear emulsion Kodak NTB-3 and stored in a cold dark box (at $4^{\circ}C$) for 3 weeks. After exposure, the samples were developed with Kodak D-19 and stained with uranyl acetate and lead citrate. Routine observation was made with Hitachi HU-11E electron microsocope. The results of the observation were as followings: 1. It was found that the labeled dbcAMP penetrated the egg plasma membrane and dispersed at random in the cytoplasm. 2. It was also observed that most of the labeled dbcAMP was attached to microfibrillar lattices portion of the oocyte cytoplasm. There fore, it is presumed that the receptor of the dbcAMP is localized in the microfibrillar lattices of the oocyte. 3. It also seems that some other cell organells such as mitochondria, Golgi complex, cortical granules are not directly related to the action of the dbcAMP. 4. The labeled dbcAMP was neither observed in the membrane nor in the nucleus. Therefore, it seems that there is no relationship between the concentration of dbcAMP and the nuclear membranous permeability. 5. There was no difference in number of dbcAMP particles when oocytes were cultured for 6 hours and 19 hours. 6. However, it was observed that, in same of the oocytes suppressed in germinal vesicle by dbcAMP for 19 hours, cell organells were moved and concentrated to a small portion of the cytoplasm, and that the morphology of the organells greatly changed to an abnormal. form. Therefore, it is supposed that those oocytes were in the process of degeneration. From the above results, it is expected that dbcAMP penetrated the egg membrane and was bound to the receptor which seems to be located in the microfibrillar lattiees portion, and that this dbcAMP-receptor complex inhibited some enzyme system of the oocytes which are essential for the germinal vesicle breakdown.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.3
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• pp.235-243
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• 2000

Objective: Our present studies were conducted to examine more effective isolating method of preantral follicles from mouse ovaries. Methods: ICR mice (3-6 weeks old) were sacrificed through cervical dislocation and their ovaries were removed and put into watch glasses containing Hams F-10 supplemented with 10% fetal bovine serum (FBS). Preantral follicles were isolated by three different methods; 1) enzymatical method and 2) mincing method, and 3) scraping method. Enzymatical method was carried out as following. Ovaries were bisected with a pair of fine 30G needles. Bisected ovaries were incubated at $37^{\circ}C$ and 5% $CO_2$ incubator in 2-well dish containing Hams F-10 supplemented with collagenase 600 lU/ml and DNAse 20 lU/ml. After 20 min., follicles were isolated by repeated pipetting. Isolated preantral follicles were collected, and the remnant of tissues was placed in incubator and previous procedure was repeated. Mincing method was carried out with a pair of fine 30G needles attached to 1 ml syringes and minced ovary. Scraping method was carried out with a pair of fine 30G needles and scratched to surface of ovary. The differences between isolating methods were analyzed using Student's t-test and Chi-square. Results were considered statistically significant when ${\rho}$ value was less than 0.05. Results: In handling time, mincing or scraping method ($28{\pm}3.42$ min or $16{\pm}1.58$ min) were significantly (p<0.00001) shorter than enzymatical method ($72{\pm}1.69$ min), and scraping method was significantly (p<0.01) shorter than mincing method. Total number of isolated follicles was significantly (p<0.0001) higher in enzymatical method ($49.8{\pm}3.91$) than in mincing or scraping method ($25.3{\pm}2.33$ or $20.5{\pm}1.75$). Isolated follicles in ${\leq}$90${\mu}m$ were significantly (p<0.005) higher in enzymatical method ($15{\pm}1.71$) than in mincing or scraping method ($7.8{\pm}0.98$ or $8.1{\pm}1.31$). In 91~130 ${\mu}m$, isolated follicles were significantly (p<0.0005) higher in enzymatical method ($33{\pm}3.27$) than in mincing or scraping method ($16.3{\pm}1.82$ or $10.7{\pm}1.38$). In ${\geq}$ 131 ${\mu}m$, isolated follicles were not significantly differences between all groups. In equal sizes, the rate of isolated follicles in ${\leq}$ 90 ${\mu}m$ was highest in scraping method (39.6% vs. enzymatical method: 30.1%, p<0.05; mincing method: 30.9%, p=0.11719, NS). Rate of follicles in $91{\sim}130$ ${\mu}m$ was significantly (p<0.05) lower in scraping method (52.7%) than in enzymatical or mincing method (66.3% or 64.5%). Rate of follicles in ${\geq}$131 ${\mu}m$ was highest in scraping method (8.3% vs. enzymatical or scraping method: 3.6%, p<0.05 or 4.6%, p=0.19053, NS). Conclusions: This study suggests that scraping method is simple and useful for isolation of preantral follicles, because this method reduced handling time and recovered enough follicles. The recovered rate of isolated follicles in diameter of 91 ~ 130 ${\mu}m$ was highest in all methods.

• Applied Microscopy
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• v.37 no.1
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• pp.11-21
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• 2007

Saengshik which is food not to apply heat is expected to have good effect on the body because metabolic enzymes are preserved in it. In fact, despite of enlargement of saengshik market, the researches on the effect of saengshik are insufficient. So, the aim of this study was to investigate the protective effect of saengshik against $CCl_4-inducing$ hepatotoxicity in ICR mice. As the result of serum biochemical parameters, AST (SGOT) and ALT (SGPT) were significantly decreased at the experimental group treated with saengshik and $CCl_4$ as compared with experimental group 1 treated with $CCl_4$ only (p<0.01). ALP was significantly higher at experimental group 2 compared with control and experimental group 1 (p<0.01). In histological study, experimental group 1 treated with $CCl_4$ only showed severe necrosis all over the liver at the gross observation, and showed severe perivenular necrosis and individual hepatocytic degeneration. However experimental group 2 was not nearly different from control group in appearance and showed mild perivenular necrosis and dominant sever neutrophil infiltration surrounding central veins. The result of this study showed the protective effect against $CCl_4-inducing$ hepatotoxicity and suggest the possibility of the functional health food. However, further studies about the effect of saengshik may be needed.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.79-89
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• 2001

Steroid hormones control the expression of many cellular regulators, and a role thor estrogen in mouse oocytes has been well documented. The preovulatory $E_2$increment is generally accepted as the endocrine process regulating induction of in vivo oocyte maturation To address whether the activity of the T-type $Ca^{2+}$ channel is altered by 17 beta-estradiol ( $E_2$), we examined the actions of $E_2$on the calcium channel of mouse oocytes and early embryos. Oocrtes were collected from the oviduct of mice treated with pregnant mare's serum gonadotropin (PMSG) and human choronic gonadotropin (hCG). Whole cell voltage clamp technique and confocal microscopy were used to examine that $E_2$increase intracellular $Ca^{2+}$ concentration ([C $a^{2+}$]$_{i}$ ) via voltage dependent $Ca^{2+}$ channel (VDC) and estrogen receptor (FSR), and $E_2$concentration by the use of radioimmunoassay (RIA) were examined in mouse. The results obtained were as follows: The peak of $Ca^{2+}$ current induced by $E_2$increased 122% to 1.50$\pm$0.03 nA from 1.23$\pm$0.21 nA (n=15) in the presence of 5 mM extracellular $Ca^{2+}$ concentration ([C $a^{2+}$]$_{o}$ ). The increased $Ca^{2+}$ current was temporally associated with $Ca^{2+}$ transients. The intracellular $Ca^{2+}$ level increased 207%~30 s following the addition of 1${\mu}{\textrm}{m}$ $E_2$(relative fluorescence intensity: 836.4$\pm$131.2 for control, n=10, 1736.4$\pm$192.0 in the presence of $E_2$, n=10). $E_2$increased amplitude of $Ca^{2+}$ current and [C $a^{2+}$]$_{i}$ . $E_2$-induced $Ca^{2+}$ current and $E_2$concentration in blood were showed difference on the stage of embryo. These results suggest that $E_2$modulate $Ca^{2+}$ channel to increase $Ca^{2+}$ influx.$Ca^{2+}$ influx.

• Journal of the Korean Society of Food Science and Nutrition
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• v.41 no.7
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• pp.950-956
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• 2012

Salicornia herbacea L. is an annual herb that grows in salt marshes and salt fields along the seashores. It is also commonly used as a folk remedy in Korea. The objective of this study was to investigate the beneficial effects of Salicorrnia herbacea L. (SH) hot water extract on obesity. Five-week old male Sprague-Dawley rats (n=36) were divided into two groups and provided either a normal fat diet (11.5% fat from kcal) or a high fat diet (40.5% fat from kcal) for 6 weeks to induce obesity. Then, rats were blocked into six groups of six mice each and provided either a diet containing SH (0.5% of diet; g/kg) or a normal diet for another 6 weeks. Final body weights were significantly reduced when rats were fed SH among the high fat diet groups (HNS and HHS). Serum triglyceride concentrations significantly decreased in every group provided SH. HDL-cholesterol concentrations significantly increased in SH-fed groups among the high fat diet groups. Further, atherogenic index significantly decreased when rats were fed SH diet (HHS). There were no differences in LDL-cholesterol between the high fat diet groups, and glucose concentrations decreased when rats were fed SH diet (HNS). These results indicate that dietary intake of Salicornia herbacea L. hot water extract might have beneficial effects on obesity by reducing body weight, fat weights, and improving blood lipid profile.

• Journal of the Korean Society of Food Science and Nutrition
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• v.43 no.3
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• pp.341-348
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• 2014

We investigated the anti-obesity and anti-hyperlipidemic effects of extracts of two roasted coffee beans (Vietnam robusta and Ethiopia mocha sidamo G2) and fermented coffee beans with Monascus rubber mycelium (MR) by solid-state culture. C57B/L6 mice were divided into seven groups: normal diet (ND) group, high fat diet (HFD) group, and HFD groups with hot water extracts from Vietnam robusta coffee beans (HFD-VR), MR-fermented Vietnam robusta coffee beans (HFD-VR-MR), MR-fermented Vietnam robusta coffee beans with 10% brown rice (HFD-VR-MR-BR10), Ethiopia mocha sidamo G2 coffee beans (HFD-ES), and MR-fermented Ethiopia mocha sidamo G2 coffee beans (HFD-ES-MR). After 6 weeks, body weight gain and food efficiency ratio were higher in the HFD group, but significantly reduced in the coffee extracts-fed groups. The HFD-ES-MR group showed greater body weight reduction than the HFD-ES group. The serum triglyceride, total cholesterol, and LDL-cholesterol levels as well as the atherogenic index and cardiac risk factor all tended to decrease in groups fed Vietnam robusta coffee extracts compared to the HFD group. These results suggest that Vietnam robusta and Ethiopia mocha sidamo G2 may be used to make functional coffee beverages with anti-obesity and anti-hyperlipidemic activities.